Increased Procoagulant Activity Research Articles

#855 Cyclophilin D in cholesterol crystal-induced acute kidney injury

Abstract Background and Aims Cholesterol crystal embolism (CCE) occurs when cholesterol crystal (CC) clots move from an atherosclerotic plaque in a large artery to a smaller artery, leading to obstruction, ischemia, and organ failure. CCE directly impairs blood flow by provoking a local inflammatory response and triggering arterial thrombus formation, consisting of collagen and fibrin mesh, platelet and neutrophil activation, and extracellular DNA trap formation. Activated platelets form a procoagulant surface that supports adhesion to collagen and fibrin mesh, promoting thrombus growth and the recruitment of immune cells. CCE often targets kidney tissue, leading to thrombotic angiopathy, acute loss of glomerular filtration rate (GFR), and necrosis in tubular cells. Cyclophilin D (CypD) plays a central role in regulating platelet procoagulant activity by triggering calcium bursts and mitochondrial depolarization. While the increased number of procoagulant platelets contributes to thrombosis, CypD-mediated procoagulant pathomechanisms in the kidney during CCE remain unknown. In our study presented here, we hypothesized that CypD deficiency might attenuate procoagulant platelet activity, thereby inhibiting CCE-induced kidney infarction and organ injury. Method 6-week-old wild-type (WT) and CypD-/- mice were administered 10 mg/kg CC through injections into the left kidney arteries in vivo. Kidneys were collected 24 hours later, and kidney function, infarct size, necroinflammation and kidney injury were evaluated using GFR measurements, TTC colorimetric assay, immunohistochemistry, and immunofluorescence microscopy. For in vitro studies, whole blood samples were isolated from WT and CypD-/- mice and perfused on a collagen-coated surface in the presence or absence of CC under arterial shear conditions of 1000s-1. Results After CC injection in mice, CypD deficiency ameliorated immunothrombosis, acute kidney injury, and organ failure. CypD-/- mice were protected from the sudden drop in GFR, i.e., acute kidney injury, which resulted from the reduced number of arteries obstructed by crystal clots. Kidney infarct size consistently decreased in CypD-/- mice. CypD-/- mice displayed reduced neutrophil recruitment to thrombotic microangiopathic lesions, indicating diminished immunothrombosis. Preemptive injection of the CypD inhibitor cyclosporin A could prevent CC-mediated vessel obstruction and infarct size, thereby improving kidney function. Interestingly, WT platelets showed increased platelet adhesion and phosphatidylserine (PS) exposure on a collagen-coated surface in the presence of CC under shear conditions in whole blood, suggesting increased procoagulant activity, which was strongly reduced in CypD-/- platelets. Conclusion During CCE-mediated kidney injury, CypD-mediated platelet activation and consequent procoagulant activity accelerate ischemic tissue necrosis and the recruitment of neutrophils. Inhibition of CypD-mediated signaling could be a potential therapeutic approach to inhibit acute kidney injury in patients with CCE.

Thrombosis Caused By a Novel Coagulation Factor IX Mutation (FIX Shanghai II)

Most thrombophilia patients are caused by insufficient suppression of coagulation reactions, which could be either mediated by deficiencies of protein C, protein S,antithrombin, or inhibition-resistant coagulation factor mutants, such as Factor V Leiden and prothrombin Yukuhashi. The thrombosis caused by coagulation factor mutants with increased procoagulant activity is very rare and so far only two Factor IX (FIX) variants involving residue Arg384, FIX Padua (p.Arg384Leu) and Shanghai (p.Arg384Gln), which increased FIX activity by up to 8 folds, had been reported to be associated with the incidence of thrombosis. In a young male venous thrombosis patient we had identified a novel F9 gene missense mutation c.1018G>A, resulting in p. Glu340Lys substitution (designated as FIX Shanghai II). The patient's APTT time was 28.9 seconds (normal reference 22.3~38.7s), and his PT was also within the normal reference range (11.5 seconds, normal reference 10.0~16.0s). Although the patient had the FIX: Ag comparable to the normal reference, 128.20±6.07%, the his FIX:C was around 201.69±10.38% of normal control (normal reference 50~150%). Thrombin generation assay (TGA) showed that compared with normal control, the patient had a shorter Lag time (2.67 vs 3.33 min), elevated ETP (1649.43 vs 1311.62 nM*min), and higher Peak value (387.34 vs 200.86 nM), indicating increased global coagulation activity of the patient. The activity of protein C, protein S, and antithrombin were all within the normal reference ranges and the patient was negative for antiphospholipid antibodies. Consistent with the patient's coagulation laboratory manifestations, the purified recombinant FIX Glu340Lys mutant showed 1.82 folds higher procoagulant activity than wild type FIX as determined by FIX:C/FIX:Ag ratio. The recombinant FIX Shanghai II mutant can be converted into FIXa by activated factor XI (FXIa) in a similar fashion as FIX WT. The kinetic study of FIXa-catalyzed FX activation showed that p. Glu340Lys mutation led to accelerated FX activation by FIXa. The p.Glu340Lys mutation also enhanced the procoagulant activity of previously reported thrombosis-causing FIX mutants, FIX Shanghai and Padua, in a similar fashion as it did on FIX WT. The FIX:C/FIX:Ag ratio of double mutants containing Glu340Lys and Arg384Gln (FIX Shanghai) or Arg384Leu (FIX Padua) was about twice of FIX Shanghai or Padua alone, 11.48- and 22.53-folds vs 6.16- and 8.13- folds higher than FIX WT respectively.

Rare Case of Chronic Limb-Threatening Ischemia in a 32-Year-Old Patient with Nephrotic Syndrome: A Case Report

AbstractChronic limb-threatening ischemia represents the end stage of peripheral artery disease (PAD), primarily affecting individuals over 60 years old. While quite rare, nephrotic syndrome (NS) is recognized for increasing the susceptibility to arterial thromboembolism (ATE). A 32-year-old male complained of resting pain in his left leg and pain after walking 50 meters with his right leg. He had a 9-year history of NS confirmed through biopsy and was on a daily regimen of 2 × 360 mg mycophenolic acid and 1 × 8 mg methylprednisolone. He had no history of hypertension, diabetes, or smoking. Atrophy and ulcers were observed on his left leg. Laboratory tests revealed elevated D-dimer and borderline high cholesterol levels. The right ankle-brachial index was 0.5, and for the left, it was 0.33. Computed tomography angiography identified occlusion in the left external iliac artery and right superficial femoral artery (SFA). The patient underwent percutaneous transluminal angioplasty with a plain balloon on both legs and an additional drug-eluting stent on the left SFA. He was discharged on rivaroxaban, clopidogrel, aspirin, simvastatin, mycophenolic acid, and methylprednisolone, with no complaints. The mechanism behind NS-caused ATE remains unclear, although it is associated with the loss of anticoagulants in urine, increased procoagulant activity, altered fibrinolytic systems, thrombocytosis, and enhanced platelet activation. Prolonged corticosteroid therapy in NS management also amplifies the risk of thromboembolism by promoting a hypercoagulable state. We suspected NS and the prolonged use of corticosteroids as risk factors for ATE, manifested as PAD in our patient. While optimal NS therapy may reduce the risk of PAD, prolonged corticosteroid use should be closely monitored.

Novel Strategy to Combat the Procoagulant Phenotype in Heparin-Induced Thrombocytopenia Using 12-LOX Inhibition.

Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Abstract 242: A Novel Strategy To Combat The Procoagulant Phenotype In Immune Thrombotic Thrombocytopenia Using 12-LOX Inhibition

The risk of stroke or other thrombotic complications following vaccination of SARS-CoV-2 has been extensively reported since widespread vaccination efforts began in early 2021. This disorder, termed vaccine-induced immune thrombotic thrombocytopenia (VITT), has remarkable overlap in clinical manifestations to that of heparin-induced thrombocytopenia (HIT). HIT and VITT are pathologies caused by IgG immune complexes that activate platelets via Fc Receptor for IgG IIA (FcγRIIA), which are collectively categorized as immune thrombotic thrombocytopenia (ITT) disorders. Thrombotic events remain high following the standard of care treatment with concurrent risk of bleeding complications. This study sought to investigate a novel approach to treatment of ITT. Recent reports demonstrate increased procoagulant activity in ITT. However, these reports required ex vivo analysis, and in vivo relevance remains unclear. Using human and mouse model systems, we investigated the cooperativity of protease activated receptors (PARs) and FcγRIIA in ITT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4 +/+ or IIA-Par4 -/- , respectively) with a well-established model IgG immune complex (αCD9). IIA-Par4 +/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4 -/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in ITT in vivo . In human platelets, we observed that PAR1, PAR4, and FcγRIIA each contributed to the procoagulant phenotype, via receptor-specific increases in cytosolic Ca 2+ . As inhibition of 12(S)-lipoxygenase has been reported to decrease platelet activation downstream of FcγRIIA and PAR4, we used inhibitor VLX-1005 and observed significant reduction in both platelet procoagulant phenotype ex vivo , and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo . These results extend our understanding of ITT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in ITT.

Temozolomide and Lomustine Induce Tissue Factor Expression and Procoagulant Activity in Glioblastoma Cells In Vitro

Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.

Endothelial Cell Activation Enhances Thromboinflammation in Vitt

Aim: Vaccine induced thrombosis thrombocytopenia (VITT) syndrome is a rare complication of ChAdOx1 vaccination for COVID19. VITT shares similarities with heparin induced thrombocytopenia (HIT). In both VITT and HIT, patients develop antibodies against platelet factor 4 (PF4) which promote platelet activation and thrombosis. Involvement of the endothelium in the pathogenic process has been described for HIT. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface of endothelial cells leads to their activation and increased procoagulant activity (Madeeva, J Thromb Haemost, 2016). However, the contribution of the endothelium to VITT has not been described. Here, we evaluate the effect of VITT plasma or serum on endothelial cell activation and thromboinflammation using an endothelialized biochip. Method: Serum and plasma samples were received from patients fulfilling clinical criteria of VITT including venous and/or arterial thrombosis, thrombocytopenia <150 x 10^3/uL, D-dimer >5-fold of upper limit of normal within 4-42 days after 1st dose of ChAdOx1 vaccine (termed "Clinical VITT") and from healthy, age and sex matched, ChAdOx1 vaccinated individuals within the same timeframe from vaccination (termed "Vaccine controls"). PF4 ELISA and VITT functional assays were performed on all samples (Lee, Blood Adv, 2022). Human umbilical vein endothelial cells (HUVECs) were treated with heat inactivated serum or plasma in the presence of peptide GPRP. Polydimethylsiloxane microfluidic straight channels were seeded with HUVECs (Endochip) and treated with VITT or control serum and compared with TNF alpha treatment. The endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tissue factor (TF) and von Willebrand factor (vWF) were measured by immunostaining in the presence or absence of native platelet factor 4 (PF4). In some experiments HUVECs were treated with ChAdOx1 vaccine or vehicle for 48-120h. Thromboinflammation was measured by fluorescence area of antibody-stained platelets, neutrophils and fibrin on the Endochip after perfusion of recalcified blood from HIT antibody-reactive healthy volunteers (Morel-Kopp, J Thromb Haemost, 2016) at a shear rate of 100s-1. Results: Samples from 10 patients presenting with clinically confirmed VITT were paired with samples from 10 vaccinated individuals. Clinical VITT samples were separated into negative and positive for both PF4 ELISA and VITT functional assay (defined as VITT ELISA positive and VITT ELISA negative, Panel A). Endothelial cell surface expression of P-selectin was increased after 12 h incubation with 25% v/v VITT serum or plasma compared with samples from AZ vaccinated non-VITT individuals (median integrated intensity/cell 8065 vs 3515 a.u, p=0.03). The expression of TF was significantly increased by addition of PF4 to the VITT plasma (median integrated intensity/cell 42038 vs 14881 a.u., p=0.01 by Mann Whitney test). ChAdOx1 treatment of HUVECs at 1:1000 dilution induced the expression of spike protein but did not significantly enhance thromboinflammation compared with media, as assessed by the perfusion of whole blood through the Endochip. Treatment of the Endochip with VITT serum, without the addition of PF4, significantly increased the adhesion of platelets compared with control serum (median 2.2-fold, p=0.002), neutrophils (2.5-fold, p=0.003) and fibrin (5.4-fold, p=0.002 by Wilcoxon paired samples test (Panel B). There was no difference in the thromboinflammatory response between ELISA negative and positive group (Panel B). Conclusion: Endothelial inflammation contributes to the prothrombotic tendency in VITT in an in vitro model of thromboinflammation. VITT serum induces the endothelial expression of P-selectin and TF. Endothelial inflammation may explain thrombotic events in cerebral or splanchnic veins of vaccinated individuals without detectable anti-PF4 antibodies by ELISA or platelet-based VITT functional assays. Endothelialised biochips have diagnostic potential for detecting immune prothrombotic tendencies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

THE STATE OF SOME INDICATORS OF HEMOSTASIS IN RELATIVELY HEALTHY PEOPLE AND PATIENTS WITH ISCHEMIC STROKE, TAKING INTO ACCOUNT GENDER DIFFERENCES

Goal. To study some indicators of hemostasis in a comparative aspect in patients with ischemic stroke and healthy people, taking into account gender differences.&#x0D; Materials and methods. In the course of the study, the hemostasis indices of 160 people were studied. All the examined were divided into two subgroups, the first consisted of 100 donors, 50 men and women each, who did not have acute and chronic diseases. The second group included the study of analogical indicators in 60 people, 30 men and women, with an established diagnosis of acute ischemic cerebral circulation disorder.&#x0D; Results. As the results show, differences in hemostasis indicators were found, taking into account gender differences, and disorders in the hemostasis system play an important role in the pathogenesis of ischemic stroke. The obtained results suggest that as a result of the development of endothelial tissue dysfunction, the level of endothelin increased, which led to pronounced vascular spasm and increased procoagulant activity of the blood.&#x0D; Conclusion. Based on the conducted research, it was found that in men and women at the time of admission with ischemic stroke, in comparison with the results obtained from donors without established pathology, changes in red blood parameters and rheological properties of blood are observed in the blood. It should be noted that in the group of female patients, changes in the rheological properties of blood were less pronounced than in the group of male patients.

The procoagulant effects of extracellular vesicles derived from hypoxic endothelial cells can be selectively inhibited by inorganic nitrite

BackgroundExtracellular vesicles (EVs) derived from endothelial cells are elevated in cardiovascular disease and promote inflammation and coagulation. Hypoxia is often a key feature and is itself a potent stimulator of increased EV production. Inorganic nitrite (NO2−) has beneficial and protective effects that are enhanced in hypoxia. ObjectivesInvestigate the impact of hypoxia on the functional capacity of EV derived from endothelial cells under hypoxia, and assess whether pre-treatment of endothelial cells with NO2− can alter EV function. MethodsDifferential ultracentrifugation was used to isolate EV from the cultured endothelial cell line HECV (CEV), and from primary human umbilical cord derived endothelial cells (PEV), with time-resolved fluorescence used to assess EV protein composition. Clot formation was induced by thrombin and calcium in two assays; using an Alexa Fluor 594 human fibrinogen conjugate assay and standard turbidometry. Platelet aggregation was determined using multiple electrode aggregometry. Scanning electron microscopy was used to visualise fibrin clots. ResultsHypoxia exposure (1% O2) significantly increased CEV production in comparison to normoxia (21% O2) (1825 ± 72 EVs/cell vs 117 ± 9 EVs/cell, p < 0.001, respectively) but had no effect on CEV mean size (221 ± 6 nm vs 203 ± 4 nm, p > 0.05). Hypoxia-derived PEVs contained significantly more tissue factor than normoxia-derived EVs (Relative Fluorescence Units (RFU) = 7666 ± 1698 vs 5958 ± 1644, p < 0.001, respectively) and less tissue factor pathway inhibitor (RFU = 9799 ± 2353 vs 19723 ± 2698, p < 0.05). Hypoxia significantly increased CEV induced fibrin polymer formation compared to normoxia (% area = 46.98 ± 0.97 vs 36.36 ± 0.72, p < 0.05). Pre-treatment of endothelial cells with NO2− in hypoxia abrogated this effect (% area = 15.70 ± 1.99, p < 0.001). Hypoxia derived CEV non-significantly increased the maximum clot formed, shortened time to max clot, and increased time to clot lysis by turbidometry. ADP-mediated platelet aggregation was significantly elevated with PEV derived from hypoxia compared to normoxia (888.0 ± 32.2 AU*min vs 671.5.2 ± 28.3 AU*min, p < 0.01). This was abrogated by pre-treatment of hypoxic endothelial cells with NO2− (716.5 ± 744.3 AU*min, p < 0.001). ConclusionsHypoxia-derived PEVs and CEVs exhibit increased procoagulant activity compared to normoxia-derived EVs, which we confirm to be mediated by an imbalance of TF/TFPI. Pre-treatment of endothelial cells with NO2− reduces the pro-coagulant activity of EVs via a mechanism that is Hypoxia-inducible factor 1 (HIF-1) dependent, but independent of TF/TFPI.

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

Titanium dioxide nanoparticles enhance thrombosis through triggering the phosphatidylserine exposure and procoagulant activation of red blood cells

BackgroundExpanding biomedical application of anatase titanium dioxide (TiO2) nanoparticles (NPs) is raising the public concern on its potential health hazards. Here, we demonstrated that TiO2 NPs can increase phosphatidylserine (PS) exposure and procoagulant activity of red blood cells (RBCs), which may contribute to thrombosis.ResultsWe conducted in vitro studies using RBCs freshly isolated from healthy male volunteers. TiO2 NPs exposure (≦ 25 μg/mL) induced PS exposure and microvesicles (MV) generation accompanied by morphological changes of RBCs. While ROS generation was not observed following the exposure to TiO2 NPs, intracellular calcium increased and caspase-3 was activated, which up-regulated scramblase activity, leading to PS exposure. RBCs exposed to TiO2 NPs could increase procoagulant activity as measured by accelerated thrombin generation, and enhancement of RBC-endothelial cells adhesion and RBC-RBC aggregation. Confirming the procoagulant activation of RBC in vitro, exposure to TiO2 NPs (2 mg/kg intravenously injection) in rats increased thrombus formation in the venous thrombosis model.ConclusionCollectively, these results suggest that anatase TiO2 NPs may harbor prothrombotic risks by promoting the procoagulant activity of RBCs, which needs attention for its biomedical application.

Subcutaneous engineered factor VIIa marzeptacog alfa (activated) in hemophilia with inhibitors: Phase 2 trial of pharmacokinetics, pharmacodynamics, efficacy, and safety

BackgroundMarzeptacog alfa (activated) (MarzAA), a novel recombinant activated human factor VII (FVIIa) variant, was developed to provide increased procoagulant activity, subcutaneous (SC) administration, and longer duration of action in people with hemophilia. ObjectivesTo investigate if daily SC administration of MarzAA in subjects with inhibitors can provide effective prophylaxis. MethodsThis multicenter, open‐label phase 2 trial (NCT03407651) enrolled men with severe congenital hemophilia with an inhibitor. All subjects had a baseline annualized bleeding rate (ABR) of ≥12 events/year. Subjects received a single 18 μg/kg intravenous dose of MarzAA to measure 24‐hour pharmacokinetics (PK) and pharmacodynamics (PD), single 30 μg/kg SC dose to measure 48‐hour PK/PD, then daily SC 30 μg/kg MarzAA for 50 days. If spontaneous bleeding occurred, the dose was sequentially escalated to 60, 90, or 120 μg/kg, with 50 days at the final effective dose without spontaneous bleeding to proceed to a 30‐day follow‐up. The primary end point was reduction in ABR. Secondary end points were safety, tolerability, and antidrug antibody (ADA) formation. ResultsIn the 11 subjects, the mean ABR significantly reduced from 19.8 to 1.6, and the mean proportion of days with bleeding significantly reduced from 12.3% to 0.8%. Of a total of 517 SC doses, six injection site reactions in two subjects were reported. No ADAs were detected. One fatal unrelated serious adverse event occurred: intracerebral hemorrhage due to untreated hypertension. ConclusionsThe data demonstrated that MarzAA was highly efficacious for prophylactic treatment in patients with inhibitors by significantly decreasing bleed frequency and duration of bleeding episodes.

“MORE THAN IT MEETS THE EYE” – AN ATYPICAL CASE OF DENGUE AND ENTERIC FEVER COINFECTION COMPLICATED BY PULMONARY EMBOLISM

At a time when infectious diseases , bacterial , viral and fungal are yielding to stratagems such as immunization and antimicrobial agents, dengue fever continues to pose major clinical , social and epidemiological challenges both in India and Worldwide. Dengue is a mosquito born viral disease by the vector Aedes aegyptii or Aedes albopictus during the taking of a blood meal. Dengue infection can be either asymptomatic, or progress to involve haemorrhagic manifestations with shock.[5] Enteric fever is a systemic disease characterized by fever and abdominal pain caused by dissemination of Salmonella typhi or paratyphi A, B or C. Fever is documented at presentation in more than 75% cases and typically prolonged and continuing up to 4weeks if untreated. Here we present an Atypical case of Dengue and Enteric fever coinfection in a young male where the unusual and ever changing clinical manifestation posed a nerve wrecking challenge in the management of this case. Thrombotic events have not been extensively reported, despite the wide range of increased procoagulant activity during illness.[6,7] And the peculiarity in this case was the severe dehydration Leading to a thrombophilic state which led to the dichotomy of whether to use Anticoagulants in the phase of severe dengue fever leading to very low platelet count which predisposes to spontaneous bleeding.

Safety, pharmacokinetics, and pharmacodynamics of a next‐generation subcutaneously administered coagulation factor IX variant, dalcinonacog alfa, in previously treated hemophilia B patients

BackgroundDalcinonacog alfa (DalcA), a next‐generation, recombinant human factor IX (FIX) variant, was developed using a rational design approach for increased procoagulant activity and longer duration of action to be administered subcutaneously (SC) for prophylaxis of hemophilia B bleeding episodes. ObjectivesTo investigate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of DalcA. MethodsThis multicenter, phase 1/2a study (NCT03186677) was conducted in 11 males aged 12 to 65 years with severe hemophilia B. In cohort 1, subjects received intravenous (IV) 75 IU/kg BeneFIX and DalcA. Cohorts 2 and 3 had DalcA IV 75 IU/kg and SC 75 IU/kg or 150 IU/kg. Cohort 4 was omitted. Cohort 5 received daily SC 150 IU/kg DalcA for 6 days and cohort 6 received IV 75 IU/kg and daily SC 150 IU/kg DalcA for 9 days. Blood sampling was performed for chemistry, hematology, PK, PD, and anti‐drug antibody measurement. Subjects were monitored for safety endpoints for 30 days postdosing. ResultsDalcA demonstrated a 24‐fold greater potency over BeneFIX and longer mean residence time (33.8 h). SC bioavailability 8.2% to 20.3%, beta half‐life 53.9 to 106.9 h and Tmax 24 to 48 h. A median 15.7% FIX activity level (interquartile range, 14.9%‐16.6%) was reached after 6 daily doses. Neutralizing antibodies to ISU304, but not wild‐type FIX, occurred in two cousins. ConclusionsThe data demonstrated that DalcA achieved protective FIX activity levels between 11% and 18%, corresponding to a reduced chance of spontaneous bleeds. Based on the results, a phase 2b trial to assess the safety and efficacy of 28 daily SC doses of DalcA was performed.

Pathophysiological justification of the use of photodynamic therapy in the initial stage of squamous cell skin cancer

The work is based on an analysis of the course of the disease in 185 patients with squamous cell skin cancer. The study program included studies in three major groups. The first included 74 (40%) patients who underwent combined treatment: an operation involving widespread excision of a tumor under intravenous anesthesia + photodynamic therapy (group 1). The second group was 111 (60%) patients with squamous cell carcinoma of the skin who received only operative treatment. To control and compare the obtained laboratory indicators, a second group was created, which included patients without established pathology. This group included 17 people who voluntarily agreed to conduct the study. The study traced the dynamics of changes in hemostasis scores and metalloproteinases in the two groups being compared. In the group of patients with squamous cell skin cancer, there was a statistically significant decrease in the metalloproteinase-1 inhibitor index, which led to an increase in all metalloproteinase-2, -7 and -9 indicators. Performing surgical treatment did not lead to correction of these indicators, both in the near and distant periods after treatment. The mechanism of influence of photodynamic therapy in squamous cell skin cancer can be characterized as follows: there is a decrease in the inhibitor of metalloproteinase-1, which leads to an increase in matrix metalloproteinases-2, -7 and -9 in the blood, as a result of damage to the endothelium of the vascular wall, which is confirmed by an increase in endothelin, this, in turn, led to the development of vasoconstriction and increased procoagulant activity The use of photodynamic therapy restores the index of metalloproteinase-1 inhibitor, this leads to the restoration of matrix metalloproteinases-2, -7 and -9 in the blood and the prevention of damage to the vascular wall. Comprehensive treatment of patients with squamous cell skin cancer in the initial stage of the disease should include photodynamic therapy with simultaneous intravenous blood irradiation, laser tumor training, and then surgical treatment, consisting in excision of the tumor with observance of oncological principles.

ИММУННАЯ РЕГУЛЯЦИЯ СИСТЕМЫ ГЕМОСТАЗА ПРИ ВОСПАЛИТЕЛЬНЫХ ЗАБОЛЕВАНИЯХ ПАРОДОНТА У КУРСАНТОВ УФИМСКОГО ЮРИДИЧЕСКОГО ИНСТИТУТА МИНИСТЕРСТВА ВНУТРЕННИХ ДЕЛ РОССИЙСКОЙ ФЕДЕРАЦИИ

Subject. Inflammatory periodontal diseases are accompanied by violations of the body's reactivity to the introduction of the pathogen. At various stages of the immune response, Pro-inflammatory cytokines are produced, such as interleukins 1, 6 (IL-1, IL-6), tumor necrosis factor-alpha (TNF-α), and chemokines in particular IL-8. Cytokines serve as an organizing system that forms and regulates the entire complex of protective reactions of the body, provides activation of various types of cells, including white blood cells, dendritic, endothelial, epithelial cells, fibroblasts, macrophages, lymphocytes, and others. They cause endothelial activation, leading to increased permeability, increased expression of adhesive molecules, and increased procoagulant activity. The appearance of interleukins in the focus of inflammation causes the production of tissue factor, Willebrand factor, plasminogen activators, etc., which stimulate the reactions of vascular-platelet hemostasis, which activate the Pro-enzyme-enzyme cascade of blood clotting and fibrinolysis.&#x0D; The aim — to study the effect of cytokines on the hemostatic system in individuals with healthy periodontitis, chronic gingivitis, and chronic generalized periodontitis.&#x0D; Methodology. 60 cadets aged 18 to 25 years participated in the clinical study at the dental office of the medical unit No. 1 of the medical and sanitary unit of the Ministry of Internal Affairs of the Russian Federation (Ufa). The study group consisted of 45 people: 15 patients with intact periodontitis, 15 patients with moderate chronic gingivitis, and 15 patients with moderate chronic generalized periodontitis.&#x0D; Results. Correlations between IL-1β, IL-6, TNF-α, and IL-8 in gingival sulcus flushes, pockets, and hemostasis parameters were revealed.&#x0D; Conclusions. In inflammatory periodontal diseases, interleukins can inhibit fibrinolysis, leading to hypercoagulation. Increased thrombosis contributes to the progression of gingivitis to periodontitis.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations.Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay.Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells.Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Cryopreserved Platelets Retain Agonist Responsiveness and Support for Factor VIII Function

Platelet activation supports procoagulant activity through phosphatidylserine exposure, secretion of procoagulant factors, and receptor conformational change. For example, thrombin-stimulated platelets bind factor VIII (fVIII) via a macromolecular complex including oligomeric fibrin and the active αIIbβ3 receptor (Phillips et al, JTH 2004; Gilbert et al, Blood 2015). Thus, coagulation assays in which phospholipid vesicles are substituted for platelets do not fully emulate modulators of fVIII activity. Indeed, inhibition of platelet-supported fVIII activity by a panel of mAbs against the C2 domain was not correlated to inhibition of vesicle-dependent activity (Chatterjee et al, JTH 2020). An obstacle to adoption of a platelet-based assay for fVIII is the need for fresh platelets. Therefore we asked whether cryopreserved platelets might support fVIII activity similarly to fresh platelets. Apheresis platelets were mixed with cryopreservatives with or without calcium chelators, in various aliquot sizes, and frozen on various cooling media. Cryopreserved platelets were compared to non-preserved apheresis platelets with regard to agonist response and support of procoagulant activity. Cryopreservation resulted in an increase in subcellular debris and an unresponsive fraction of platelets with decreased forward scatter judged by flow cytometry. Optimized results were obtained when platelet rich plasma with 5% DMSO, in 1 mL aliquots was frozen on powdered dry ice, and stored at -150C. Purification of thawed platelets using a density gradient removed debris and decreased unresponsive platelets resulting in a forward and side scatter profile comparable to fresh platelets. We refer to these as cryopreserved platelets (CryoPlts). CryoPlts were compared to control and outdated apheresis platelets. As with fresh platelets, procoagulant activity of CryoPlts increased with thrombin receptor agonist peptides (TRAP) 1 &amp; 4 and supported a log-linear relationship between time to initial fibrin strand formation and fVIII activity over a range of 0.0001 - 1 u/mL (Fig 1). Further, the degree of inhibition of fVIII activity by mAbs ESH4 and G99 against the fVIII C2 domain, was the same on control and CryoPlts, but markedly different from inhibition in an aPTT-based inhibitor assay. In contrast, outdated apheresis platelets had increased procoagulant activity, minimal agonist response and a shallow curve with varying fVIII concentration. Flow cytometry studies with lactadherin-FITC indicated that 33 ± 14% of CryoPlts had high PS exposure, and the size of this population was minimally affected by TRAP 1+4. In contrast, the main platelet population had a small, uniform, increment in PS exposure comparable to control platelets. Surprisingly, the PS-rich platelets did not significantly affect the time to fibrin formation, confirming that the viable platelets, with limited PS exposure, provide much of the support for fVIII-related procoagulant activity. Flow cytometry indicated αIIbβ3 activation (PAC1-FITC) and α-granule release (anti-P-selectin-PE) were qualitatively intact on CryoPlts, although staining was decreased 70% for PAC1 and 57% for Psel. We also tested whether CryoPlts may be utilized for evaluating response to anti-PF4-heparin antibodies, relevant to heparin-induced thrombocytopenia (HIT). We evaluated platelet response to platelet factor 4 (PF4) and a platelet-activating anti-PF4 antibody (KKO), a combination that induces activation similar to authentic autoimmune antibodies for HIT. The non-activating PF4 antibody RTO served as a negative control. Geometric mean response, corrected for background, was normalized to response to thrombin activation. Both fresh and CryoPlts responded with increases in PAC1 (Fig 2A) and anti-Psel (Fig 2B) binding in response to KKO/PF4 compared to RTO/PF4 . This data demonstrates that the qualitative αIIbβ3 and P-selectin response to HIT-like antibodies is intact. Our results demonstrate a refined cryopreservation protocol of apheresis platelets. These platelets maintain qualitative agonist responsiveness with near-normal support for factor VIII activity, suggesting that they could be used for other platelet-based laboratory or diagnostic assays. Further, our results suggest that major procoagulant activity is provided by platelets with very limited PS exposure, an area for further investigation. Disclosures No relevant conflicts of interest to declare.

Hypercoagulability in COVID-19

COVID-19 is associated with a hypercoagulable state that may present as pulmonary thrombosis, pulmonary embolism, and venous and arterial thrombosis.Suggested pathogenesis include direct infection of the endothelial cell with subsequent endothelial cell dysfunction, leading to increased procoagulant activity, decreased anticoagulant activity and decreased fibrinolysis. The severe immune inflammatory response in the lungs with cytokine release also plays a critical role (immunothrombosis). Hypoxia has a local and systemic effect on coagulation. Various markers of this state have been described, and especially the D-dimer level (and rapid changes in the D-dimer level) as a reliable prognostic marker. It is also used as indicator for initiation of anticoagulation by some experts. Due to the pleotrophic effects of heparin, it is the anticoagulant of choice for these patients (most often low molecular weight heparin, due to decreased risk of heparin induced thrombocytopenia, ease of use). No clinical trial data is available at the time of writing (28 May 2020), and suggested guidelines of experts in different countries are discussed.

Serum concentration of selected biochemical markers of endothelial dysfunction and inflammation in patients with the varying activity of inflammatory bowel disease.

Endothelial dysfunction leads to an increased expression of cell adhesion molecules, leukocyte diapedesis, vascular smooth‑muscle tone, excessive permeability of vascular walls, and increased procoagulant activity. We investigated whether serum levels of several endothelial and platelet activation markers correlated with disease activity in patients with inflammatory bowel disease (IBD). This study included 56 patients with ulcerative colitis, 66 with Crohn disease, and 40 healthy controls. We measured the complete blood count and levels of fibrinogen, C‑reactive protein, albumin, interleukin 6, tumor necrosis factor α, E‑selectin, P‑selectin, monocyte chemoattractant protein 1 (MCP‑1), soluble CD40 ligand (sCD40L), and microparticles. There were no significant differences in the median levels of E‑selectin, P‑selectin, MCP‑1, sCD40L, and microparticles between patients with active IBD, those with inactive IBD, and healthy controls. The clinical disease activity assessed with the Mayo scale in the ulcerative‑colitis group was weakly, positively correlated with sCD40L (R = 0.32, P = 0.02), P‑selectin (R = 0.32, P = 0.02), and inflammatory marker levels. The clinical disease activity index in the Crohn disease group was positively correlated with the markers of inflammation yet not with the markers of endothelial activity. E‑selectin, P‑selectin, sCD40L, MCP‑1, and microparticle levels do not significantly differ between patients with the varying activity of IBD. However, due to the observed correlations, further studies of a larger patient group should be conducted to confirm our observations.