Procoagulant Effects Research Articles

Thein Vitroprocoagulant Effects of Standard and Extended Half-Life Recombinant Factor IX Concentrates in Patients on Prophylaxis with Emicizumab

Introduction: Emicizumab is a humanized, monoclonal, bispecific antibody that binds factor (F) FX and FIXa allowing thrombin generation in the absence of FVIII, which is used for routine treatment of patients with Hemophilia A (HA) with and without inhibitors. Plasma level of FIX will be an important limiting factor for the formation of the FX-FIXa-emicizumab ternary complex in the absence of FVIII, suggesting the potential use of FIX concentrates to regulate the procoagulant function of emicizumab and therefore, to use it as an alternative treatment in certain circumstances to stop or prevent bleedings in patients on prophylaxis with emicizumab. At the present time there are several recombinant FIX concentrates with differences in their content of activated FIX (FIXa) and in their half-life (standard or extended) that may differ in their procoagulant effects when combined with emicizumab. Aim: The aim of this study was to evaluate if there were differences in thein vitroprocoagulant effects of two recombinant FIX concentrates, one with standard half-life (rFIX Nonacog Alfa, BeneFIX®, Pfizer) and the other with extended half-life (rFIX fused to rAlbumin, Albutrepenonacog Alfa, Idelvion®, CLS Behring) in samples from patients on prophylaxis with Emicizumab. Methods: This is a prospective and transversal pilot study that was approved by the Ethics Committee from La Paz University Hospital. Two patients with haemophilia A (HA) with inhibitors in prophylaxis with emicizumab were recruited and one haemophilia B (HB) patient was included as a control for the effects of FIX. Blood samples were collected in tubes with corn trypsin inhibitor (CTI, Haematologic Technologies, USA), to block thecontact phase and to only evaluate coagulation mediated by the extrinsic pathway. Levels of FIXa in concentrates of FIX were quantified using the Spectrozyme® FIXa chromogenic substrate (LOXO) and measuring the increase in OD at 405 nm. Rotational thromboelastometry (ROTEM) was performed using whole blood activated by a low concentration of tissue factor solution (final dilution 1:50,000 of EXTEM reagent) plus recalcification. Parameters evaluated in ROTEM were CT (cloting time), defined as time until detection of a clot firmness of 2 mm; and CFT (clot formation time), defined as time between detection of a clot firmness from 2 to 20 mm. Calibrated automated thrombogram (CAT)was performed using platelet free plasma (PFP) activated by low concentration of tissue factor plus phospholipids (PPP-Reagent LOW®, Stago). Parameters evaluated in CAT were: Peak, defined as maximum thrombin concentration reached, in nM; and ETP, defined as the total amount of thrombin generated over time, in nMxmin. Results: The presence of FIXa activity assayed by a chromogenic substrate was not detectable with 20 IU of Idelvion while BeneFIX® showed a specific concentration-dependent FIX activity that was blocked with the serine protease inhibitor EGR-chloromethylketone (Figure 1). ROTEM (Figure 2) and CAT (Figure 3) results showed that the addition of increased concentrations of both concentrates of rFIX produces an enhanced procoagulant effect of Emicizumab similar to the effect produced by the addition of the bypassing agent rFVIIa (NovoSeven®, NovoNordisk). These results also showed that it is necessary three-four times higher concentration (U/dl) of Idelvion® to get similar procoagulant effects that those obtained with BeneFIX®. The higher procoagulant effects of BeneFIX® were also observed in samples of a patient with severe HB. Conclusion: Global coagulation assays suggest that increasing endogenous FIX levels with two rFIX concentrates that have different FIXa content and half-life, produce an enhanced procoagulant effect of Emicizumab opening the idea of the use of these concentrates as an alternative treatment for bleedings in patients with inhibitors on prophylaxis with Emicizumab. Further studies need to be performed to evaluate the procoagulant activity of the concomitant use of different rFIX concentrates and Emicizumab, and to assess security of this therapeutic approach. This work was supported by grants from FIS-FONDOS FEDER (PI19/00631 and P19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Fernandez-Bello: Novartis:Speakers Bureau;Stago:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Current Employment, Research Funding, Speakers Bureau;Roche:Speakers Bureau;SOBI,:Research Funding;Pfizer:Speakers Bureau.García Barcenilla:Pfizer,:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Bayer:Speakers Bureau;Novartis:Speakers Bureau;NovoNordisk:Research Funding, Speakers Bureau.Alvarez Román:Roche:Speakers Bureau;Novartis:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;Pfizer,:Research Funding, Speakers Bureau;Bayer:Consultancy;SOBI,:Consultancy, Research Funding, Speakers Bureau;Grifols:Research Funding;NovoNordisk,:Research Funding, Speakers Bureau.Martín:NovoNordisk:Speakers Bureau;SOBI:Research Funding;Pfizer:Research Funding, Speakers Bureau;Roche:Speakers Bureau;Novartis:Speakers Bureau.Rivas Pollmar:Novartis:Speakers Bureau;Roche:Speakers Bureau;Pfizer:Speakers Bureau.Canales:Roche:Honoraria;Celgene:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;Sandoz:Speakers Bureau;Sandoz:Honoraria;Janssen:Speakers Bureau;iQone:Honoraria;Sandoz:Honoraria;Gilead:Honoraria;Takeda:Speakers Bureau;Novartis:Honoraria;Karyopharm:Honoraria;Janssen:Honoraria;Takeda:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau;Sandoz:Speakers Bureau;Roche:Speakers Bureau;Karyopharm:Honoraria.Butta:Grifols:Research Funding;Novartis:Speakers Bureau;ROCHE:Research Funding, Speakers Bureau;Pfizer:Speakers Bureau;SOBI:Speakers Bureau;Takeda:Research Funding, Speakers Bureau;NovoNordisk:Speakers Bureau.Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer:Honoraria;F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer:Consultancy;Grifols, Novo Nordisk, Takeda, Sobi, Pfizer:Research Funding.

Study of the Effect of Fibrinogen, Factor XIII and Recombinant Activated Factor VII in a Model of Trauma-Induced Coagulopathy

Introduction: Trauma-induced coagulopathy (TIC) is a multifactorial condition secondary to severe trauma. In TIC, early fibrinogen (FI) replacement and low dose of recombinant activated factor VII (rFVIIa) may positively impact outcome. Factor XIII (FXIII), on the other hand, may stimulate in vitro clot formation and clot stability. We hypothesized that combination of FI, rFVIIa and FXIII might normalize clot formation more effectively than the isolated use of each concentrate in a model of TIC. Aim: Evaluation of the procoagulant effect of isolated or combined use of FI, rFVIIa and FXIII in a model of TIC. Methods: TIC in vitro model was obtained by dilution of whole blood from seven healthy controls with isotonic saline (NaCl 0.9%) (2:3 whole blood:saline ratio). FI, rFVIIa and FXIII were spiked in combination or alone until obtaining final levels of 2 g/L, 1 μg/mL and 100 IU/dL respectively. Procoagulant effects of the different concentrates or their mixtures were evaluated by Rotational Thromboelastometry (ROTEM®, Werfen) triggered using starTEM® (calcium chloride 0,2 M) and exTEM® reagent (source of tissue factor) diluted with saline up to 1:100.000 (final dilution) for a better evaluation of both the extrinsic and intrinsic pathways of coagulation. The values of clotting time (CT: time until 2 mm of amplitude, in seconds), amplitude (parameter proportional to the clot strength) at 5 minutes (A5, in mm) and clot formation time (CFT: time from CT to 20 mm of amplitude, in seconds) were evaluated. Statistical analysis of differences was performed by One-Way ANOVA test assuming no paring of data and using the Holm-Sidak's correction for multiple comparisons with a family-wise significance and confidence level of 0.01. Statistical significance was set at p< 0.05. Results/Discussion: Data are summarized in Table I and Figure 1. CT needed the combination of two of more concentrates to reach the normal range suggesting that the administration of FI alone in TIC may not be enough to restore the patients' hemostatic potential. In regard to the clot strength evaluated by A5, the addition of FXIII or rFVIIa alone or in combination did not improve the value of A5 that was only normalized by the addition of FI. This effect of FI was increased in the presence of FXIII or rFVIIa which indicated that normal levels of FI might be required for rFVIIa or FXIII to be effective emphasising the possible benefit of the combinatory therapy. Like observed in A5, the velocity of clot formation evaluated by the CFT was normalised only by the addition of FI. However, the combination of FI plus FXIII + rFVIIa had a stronger effect on CFT compared with the combination of FI + FXIII or FI + rFVIIa, indicating that the improvement of thrombin generation due to rFVIIa plus an increment of fibrin formation and net stabilization through the contribution of higher levels of FI and FXIII respectively, might provide a beneficial synergistic procoagulant effect in TIC. Conclusion: The use of FI in TIC may contribute to increase the patient's hemostatic potential but might not be enough. Combinatory therapies based on the administration of FI, rFVIIa and FXIII might be of better benefit in this setting. Ex-vivo studies using blood of patients with stablished TIC might bring new insights on the possible advantages of this combinatory therapy to design more effective protocols to treat this frequent and life-threatening acquired condition. Disclosures Canales: Sandoz: Honoraria; iQone: Honoraria; Janssen: Speakers Bureau; Janssen: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Sandoz: Speakers Bureau; Novartis: Honoraria; Takeda: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Janssen: Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Gilead: Honoraria. Butta:NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Pfizer: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Grifols: Research Funding. Alvarez Román:NovoNordisk,: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy; Grifols: Research Funding. Jiménez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria.

Complement Regulates the Procoagulant Effects of HIT Immune Complexes

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p <0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p<0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p<0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents & Royalties; Apotex: Consultancy, Research Funding.

Efficacy of pro‐ and anticoagulant strategies in plasma of patients undergoing hepatobiliary surgery

BackgroundIn vitro efficacy of pro‐ and antihemostatic drugs is profoundly different in patients with compensated cirrhosis and in those who have cirrhosis and are critically ill. ObjectivesHere we assessed the efficacy of pro‐ and anticoagulant drugs in plasma of patients undergoing hepato‐pancreato‐biliary (HPB) surgery, which is associated with unique hemostatic changes. MethodsWe performed in vitro analyses on blood samples of 60 patients undergoing HPB surgery and liver transplantation: 20 orthotopic liver transplantations, 20 partial hepatectomies, and 20 pylorus‐preserving pancreaticoduodenectomies. We performed thrombin generation experiments before and after in vitro addition of fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), recombinant factor VIIa (rFVIIa), low molecular weight heparin (LMWH), unfractionated heparin, dabigatran, and rivaroxaban. ResultsWe showed that patients undergoing HPB surgery are in a hypercoagulable state by thrombin generation testing. FFP and rFVIIa had minimal effects on thrombin generation, whereas PCC had a more pronounced procoagulant effect in patients compared with controls. Dabigatran showed a more pronounced anticoagulant effect in patients compared with controls, whereas rivaroxaban and LMWH had a decreased anticoagulant effect in patients. ConclusionWe demonstrate profoundly altered in vitro efficacy of commonly used anticoagulants, in patients undergoing HPB surgery compared with healthy controls, which may have implications for anticoagulant dosing in the early postoperative period. In the correction of perioperative bleeding complications, PCCs appear much more potent than FFP or rFVIIa, and PCCs may require conservative dosing and caution in use in patients undergoing HPB surgery.

Cerebral Infarction Secondary to Vascular Infiltration of Glioblastoma Confirmed in High-Resolution Vessel Wall MRI

Acute cerebral infarction secondary to glioblastoma is rarely reported, and its mechanism is still controversial. Vascular infiltration of malignant cell, direct mass effect and local procoagulant effect were considered as main pathophysiology. We report a case of glioblastoma-related cerebral infarction documented by high resolution vessel wall magnetic resonance imaging, which reveals concentric enhancement of arterial wall and intraluminal thrombus without direct compression. These findings suggest tumor cell infiltration of intracranial vessels is one of the important mechanisms of glioblastoma-related cerebral infarction.

Neutrophil extracellular traps exacerbate coagulation and endothelial damage in patients with essential hypertension and hyperhomocysteinemia

Patients with essential hypertension (EH) and hyperhomocysteinemia (HHCY) suffer from more increased thrombotic events than those in EH alone. However, the underlying mechanisms for this effect are not well understood. This study hypothesized that neutrophil extracellular trap (NET) releasing may be triggered by HHCY in patients in EH, thereby predisposing them to a more hypercoagulable state. Using a modified-capture enzyme-linked immunosorbent assay (ELISA) method, we observed that cell-free DNA (CF-DNA) and myeloperoxidase DNA (MPO-DNA) in patients With EH and HHCY were significantly higher. The NET formation was also positively correlated with homocysteine levels, neutrophil-lymphocyte ratio (NLR), and hypercoagulable markers (thrombin-antithrombin complex, D-dimers). Furthermore, neutrophils from patients in EH with HHCY were found to be predisposed to amplified NET release when compared to patients in EH without HHCY or CTR. Coagulation function assays showed that NETs in patients With EH and HHCY resulted in a significantly increased ability to generate thrombin and fibrin than in those in EH without HHCY or CTR. These procoagulant effects of NETs in patients With EH and HHCY were markedly inhibited (approximately 70%) by the cleavage of NETs with DNase I. Isolated NETs from patients With EH and HHCY neutrophils also exerted a strong cytotoxic effect on endothelial cells (ECs), converted them to apoptosis. This study revealed a previously unrecognized association between the hypercoagulable state and neutrophils in patients With EH and HHCY. Therefore, blocking NETs may represent a new therapeutic objective for preventing thrombosis in these patients.

Coagulation Changes during Central Hypovolemia across Seasons.

Lower body negative pressure (LBNP) application simulates hemorrhage. We investigated how seasons affect coagulation values at rest and during LBNP. Healthy participants were tested in cold (November–April) and warm (May–October) months. Following a 30-min supine period, LBNP was started at −10 mmHg and increased by −10 mmHg every five minutes until a maximum of −40 mmHg. Recovery was for 10 min. Blood was collected at baseline, end of LBNP, and end of recovery. Hemostatic profiling included standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and thrombin formation markers. Seven men (25.0 ± 3.6 years, 79.7 ± 7.8 kg weight, 182.4 ± 3.3 cm height, and 23.8 ± 2.3 kg/m2 BMI) and six women (25.0 ± 2.4 years, 61.0 ± 8.4 kg weight, 167 ± 4.7 cm height, and 21.8 ± 2.4 kg/m2 BMI) participated. Baseline levels of prothrombin (FII), tissue factor (TF) and markers for thrombin generation F1+2 and the thrombin/antithrombin complex (TAT) were higher during summer. Factor VIII, prothrombin fragment 1+2 (F1+2), TAT and the coagulation time showed significant increases during LBNP in both seasons. Some calibrated automated thrombography variables (Calibrated automated thrombography (CAT): lag, time to peak (ttPeak), peak) shifted in a procoagulant direction during LBNP in summer. Red blood cell counts (RBC), hemoglobin and white blood cell counts (WBC) decreased during LBNP. LBNP application reduced prothrombin time in winter and activated partial thromboplastin time in summer. Greater levels of FII, TF, F1+2, and TAT—a more pronounced LBNP-induced procoagulative effect, especially in CAT parameters (lag time (LT), Peak, ttPeak, Velindex)—were seen in summer. These results could have substantial medical implications.

In vitro antitumor, pro-inflammatory, and pro-coagulant activities of Megalopyge opercularis J.E. Smith hemolymph and spine venom

Contact with stinging spines venom from several Lepidoptera larvae may result in skin lesions. In Mexico, envenomation outbreaks caused by Megalopyge opercularis were reported between 2015 and 2016. The aim of this study was to identify the venomous caterpillars in Nuevo Leon, Mexico and evaluate several biological activities of their hemolymph (HEV) and spine setae (SSV) venoms. M. opercularis was identified by cytochrome oxidase subunit (COI) designed primers. HEV and SSV extracts cytotoxic activity was assessed on the L5178Y-R lymphoma cell line. For apoptotic cells number and apoptosis, cells were stained with acridine orange/ethidium bromide and validated by DNA fragmentation. Human peripheral blood mononuclear cells (hPBMC) cytokine response to the extracts was measured by the cytometric bead array assay. Extracts effect on pro-coagulation activity on human plasma was also evaluated. HEV and SSV extracts significantly inhibited (p < 0.01) up to 63% L5178Y-R tumor cell growth at 125–500 µg/mL, as compared with 43% of Vincristine. About 79% extracts-treated tumor cells death was caused by apoptosis. Extracts stimulated (p < 0.01) up to 60% proliferation of resident murine lymphocytes, upregulated IL-1β, IL-6, IL-8, and TNF-α production by hPBMC, and showed potent pro-coagulant effects. The pharmacological relevance of these venoms is discussed.

Utilising venom activity to infer dietary composition of the Kenyan horned viper (Bitis worthingtoni)

Bitis are well known for being some of the most commonly encountered and medically important snake species in all of Africa. While the majority of species possess potently anticoagulant venom, only B. worthingtoni is known to possess procoagulant venom. Although known to be the basal species within the genus, B. worthingtoni is an almost completely unstudied species with even basic dietary information lacking. This study investigated various aspects of the unique procoagulant effects of B. worthingtoni venom. Coagulation assays determined the primary procoagulant effect to be driven by Factor X activating snake venom metalloprotease toxins. In addition to acting upon the mammalian blood clotting cascade, B. worthingtoni venom was also shown to clot amphibian plasma. As previous studies have shown differences in clotting factors between amphibian and mammalian plasmas, individual enzymes in snake venoms acting on plasma clotting factors can be taxon-selective. As venoms evolve under purifying selection pressures, this suggests that the procoagulant snake venom metalloprotease toxins present in B. worthingtoni have likely been retained from a recent common ancestor shared with the related amphibian-feeding Proatheris superciliaris, and that both amphibians and mammals represent a substantial proportion of B. worthingtoni current diet. Thus, taxon-specific actions of venoms may have utility in inferring dietary composition for rare or difficult to study species. An important caveat is that to validate this hypothesis field studies investigating the dietary ecology of B. worthingtoni must be conducted, as well as further investigations of its venom composition to reconstruct the molecular evolutionary history of the toxins present.

ACE2, COVID-19 Infection, Inflammation, and Coagulopathy: Missing Pieces in the Puzzle.

Engulfed by the grave consequences of the coronavirus disease 2019 (COVID-19) pandemic, a better understanding of the unique pattern of viral invasion and virulence is of utmost importance. Angiotensin (Ang)-converting enzyme (ACE) 2 is a key component in COVID-19 infection. Expressed on cell membranes in target pulmonary and intestinal host cells, ACE2 serves as an anchor for initial viral homing, binding to COVID-19 spike-protein domains to enable viral entry into cells and subsequent replication. Viral attachment is facilitated by a multiplicity of membranal and circulating proteases that further uncover attachment loci. Inherent or acquired enhancement of membrane ACE2 expression, likely leads to a higher degree of infection and may explain the predisposition to severe disease among males, diabetics, or patients with respiratory or cardiac diseases. Additionally, once attached, viral intracellular translocation and replication leads to depletion of membranal ACE2 through degradation and shedding. ACE2 generates Ang 1-7, which serves a critical role in counterbalancing the vasoconstrictive, pro-inflammatory, and pro-coagulant effects of ACE-induced Ang II. Therefore, Ang 1-7 may decline in tissues infected by COVID-19, leading to unopposed deleterious outcomes of Ang II. This likely leads to microcirculatory derangement with endothelial damage, profound inflammation, and coagulopathy that characterize the more severe clinical manifestations of COVID-19 infection. Our understanding of COVID-ACE2 associations is incomplete, and some conceptual formulations are currently speculative, leading to controversies over issues such as the usage of ACE inhibitors or Ang-receptor blockers (ARBs). This highlights the importance of focusing on ACE2 physiology in the evaluation and management of COVID-19 disease.

Epigenetic Changes and Cardiovascular Risks among Workers Occupationally Exposed to Iron and Zinc

Introduction: Several metals such as Iron and Zinc were found at high concentration in foundry particulate matter and demonstrated to have procoagulant effects, these effects may occur through epigenetic changes of pro-inflammatory genes. So, Iron and Steel workers are at a high risk for cardiovascular diseases. Aim of work: Evaluation of the risk of thrombosis and cardiovascular diseases among individuals occupationally exposed to Iron and Zinc. Materials and methods: A descriptive cross sectional comparative study was performed on 60 persons working in Iron and Steel factory and 60 subjects used as control group with no history of occupational exposure to Fe or Zinc. All study population was subjected to personal interview with specially designed questionnaire. Serum Iron and Zinc levels together with Endogenous thrombin potential (ETP) test were measured. Assessment of methylated DNA of Endothelin-1 (EDN1) and Nitric-oxide synthase-3 (NOS3) genes were performed. Results: the current study showed highly statistically significantincrease of serum Iron, Zinc levels and percentage of ETP among exposed group when compared to the control with Mean ± SD 125.6±22.9, 110.9±19.2,168±0.5 vs 63.6±15.3, 42.5±7.2, and 102.22±12.36 respectively. There was also a significant decrease of methylated DNA of EDN1and NOS3 genes among exposed group with Mean ± SD 0.71±0.3, 0.75±0.3 versus 1.51±1.01,4.09±0.68 respectively among the control group. Serum levels of Iron and Zinc and percentage of ETP were negatively correlated with methylated DNA of NOS3 and EDN1 genes r= -0.304, r=-0.450, r=-0.259, r= -0.787, r= -0.866 and r= -0.618 respectively (p value <0.005), while positive correlation were detected between Iron and Zinc levels with ETP (r= 0.692 and r=0.625). It was found that Iron, Zinc and NOS3 are determinants for END1 while the latter is the only predictor for NOS3. Conclusion: The risk of thrombosis and cardiovascular diseases were related to occupational exposure to high levels of Iron and Zinc. So, regular wearing of good quality’s personal protective equipment, especially masks and gloves, is highly recommended to decrease exposure to these metals.

Antibody profiles comprising anti phosphatidylserine/prothrombin differently affect thrombin generation and protein C resistance in antiphospholipid antibody carriers

Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are currently not included in the laboratory work-up of antiphospholipid symdrome (APS). However, several studies indicate that aPS/PT confer additional risk for thromboembolic events when added to classical antiphospholipid (aPL) antibody panel. We aimed to study thrombin generation (TG), a test that describes hyper or hypo-coagulability, in a cohort of antiphospholipid antibody (aPL) carriers with or without aPS/PT. As oral anticoagulants interfere with TG, we performed the study in carriers of aPL antibodies not on oral anticoagulants treatment.TG in tissue factor-triggered platelet-poor plasma and its inhibition by thrombomodulin was measured with a calibrated automated thrombogram method. Data are expressed as minutes (Interquartile Range).Of 55 aPL carriers, 37 were positive and 18 were negative for aPS/PT. Lag Time 5.4 min (4.1; 7.3) vs 3.4 min (3.0;4.5) is significant longer (p < 0.0001) and time to peak 9.6 min (8.1;11) vs 7.7 min (6.8;8.8) is significantly delayed (p = 0.0011) in aPS/PT positive as compared to aPS/PT negative carriers. Endogenous Thrombin Potential (ETP), peak thrombin formation and the velocity index are lower in aPS/PT positive carriers but did not reach statistical significance. Inhibition of ETP by thrombomodulin was significantly lower (protein C resistance) in aPS/PT positive vs aPS/PT negative group (22.8%±11.5 vs 34.9%±20.4, p = 0.01).In conclusion, aPS/PT positive carriers show an anticoagulant effect in TG while they exert a procoagulant effect in response to thrombomodulin-activated protein C.

Emicizumab, the factor VIII mimetic bi-specific monoclonal antibody and its measurement in plasma.

Emicizumab, a monoclonal antibody mimicking the function of factor (F) VIII in the activation of FX by FIXa, is widely used for prophylaxis in hemophilia patients with or without inhibitors to FVIII. Although it is administered at fixed dose, its measurement could be occasionally required. In principle, the emicizumab procoagulant effect could be assessed by the one-stage assay (OSA) currently used to measure FVIII. However, the OSA for FVIII presents with limitations. Furthermore, owing to its potent FVIII-like activity, emicizumab interferes with the measurement of the inhibitor to FVIII, which is often needed in patients on emicizumab. We prepared test samples by spiking a FVIII-deficient plasma with graded amounts of emicizumab. We modified the OSA for FVIII and tested plasma samples for emicizumab concentrations. Furthermore the chromogenic assay (CA) for FVIII with bovine reagents was used to assess for the FVIII inhibitor in patients on emicizumab. Slight modification of the OSA for FVIII (i.e., higher test plasma dilution and longer coagulometer acquisition time) made the regular OSA as a reliable laboratory tool to measure emicizumab concentration as shown by the identity of the regression (observed vs. expected) lines. Furthermore, the inhibitors to FVIII in patients on emicizumab, which were negative when measured by the regular Bethesda assay, were reliably measured by the CA assay employing bovine reagents. The methods currently used to measure FVIII can be easily modified to make the general clinical laboratory able to assist clinicians when dealing with patients on emicizumab.

Complement Activation and Thrombin Generation by MBL Bound to β2-Glycoprotein I.

β2-Glycoprotein I (β2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to β2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of β2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and β2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to β2-GPI was detected on the surface of HUVECs, and colocalization of MBL with β2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because β2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the β2-GPI/MBL complex may contribute to amplify similar activities of anti-β2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.

Evaluation of systemic inflammatory and thrombotic markers of cardiovascular risk among young Indian oral tobacco users

BackgroundWhile the pro-inflammatory and pro-coagulant effects of cigarette smoking have been well described, the effect of smokeless tobacco (ST) on inflammatory and coagulation markers is still not clear. The study aimed to evaluate impact of smokeless tobacco use on systemic markers of inflammation [(TLC), neutrophil-lymphocyte ratio (NLR) (ESR), interleukin (IL) IL-1β, IL-6 and tumor necrosis factor alpha (TNFα)] and hypercoagulable state [fibrinogen and d-dimer] leading to increased cardiovascular risk in ST users as compared to non-users. Methods150 healthy young adults using oral tobacco products for at least 1 year were included in the case group and 50 age-matched non-consumers as controls. Subjects with any known chronic illness or comorbidity were excluded from the study. Blood samples were tested for TLC, NLR, ESR, IL-1β, IL-6, TNFα, fibrinogen and d-dimer. Statistical analysis was done using SPSS 17.0 software. ResultsThe baseline clinical and cardio-metabolic characteristics were comparable between the two groups. ST users had significantly elevated serum IL-6 [59.29 ± 124.69 pg/mL (n = 149) vs 8.21 ± 27.27 pg/mL (n = 47), p-value = 0.005], TNFα [77.18 ± 236.10 pg/mL (n = 149) vs 8.32 ± 9.36 pg/mL (n = 47), p-value = 0.041], fibrinogen [310.53 ± 129.05 mg/dL (n = 143) vs 282.82 ± 65.23 mg/dL (n = 42), p-value = 0.045] and d-dimer [0.28 ± 0.42 mg/L (n = 144) vs 0.17 ± 0.09 mg/L (n = 45), p-value = 0.043] levels as compared to non-users. Serum TLC, NLR, ESR and IL-1β remained unchanged in ST users and were similar to that of controls. ConclusionsChronic use of ST is associated with systemic inflammation and coagulation, which may increase the risk of athero-thrombotic cardiovascular events among ST users.

Association of endothelial and red blood cell microparticles with acute myocardial infarction in Chinese: a retrospective study.

Mounting evidence suggests that endothelial cell-derived microparticles (EMPs) and red blood cell-derived microparticles (RMPs), which have procoagulant and vasoconstriction effects, are involved in the development of vascular acute cardiovascular events. The aim of this study was to analyze the circulating levels of EMPs and RMPs in patients with acute myocardial infarction (AMI), and to explore the correlations between EMPs and RMPs and the severity of coronary artery disease. Plasma samples from 110 patients with AMI and 57 non-coronary artery disease group (nonCAD) were collected in the present study. The flow cytometry was used to measure the EMPs (CD31) and RMPs (CD235a) qualitatively and quantitatively. The levels of EMPs and RMPs in the AMI group were higher than that in the Non-CAD group, yet no significant difference was found between STEMI and non-STEMI subjects. The levels of EMPs and RMPs in multi-vessel were higher than in the single-vessel l disease. In the Thrombolysis in Myocardial Infarction (TIMI) risk assessment, the levels of EMPs in the high-risk group were higher than that in both intermediate- and low-risk group. The low-risk group had the lowest EMP levels, the difference between the groups being statistically significant (P=0.001). No significant difference in RMP levels was noted upon TIMI stratification. According to the ROC curve analysis, the areas under the curve (AUC) of EMPs and RMPs were 0.706 and 0.668, respectively. The circulating levels of EMPs and RMPs in patients with AMI are elevated, and the level of EMPs is related to the degree of coronary artery disease and the prognosis risk. The EMPs are more likely to be potential biomarkers than RMPs to provide diagnostic value for AMI.

A New Antibiotic-Loaded Sol-Gel can Prevent Bacterial Intravenous Catheter-Related Infections.

The aim of this study was to evaluate the effectiveness of a moxifloxacin-loaded organic–inorganic sol-gel (A50) by locally preventing the catheter-related bloodstream infection (CRBSI) provoked by Staphylococcus epidermidis (S. epidermidis) and the effect resulting from its hydrolytic degradation on coagulation by using a rabbit in-vivo model. A50 coating can completely inhibit growth and would locally prevent CRBSI provoked by S. epidermidis. None of the coagulation blood parameters showed a significant difference constant over time between the control catheter group and the A50-coated catheter group, despite the visible silica release resulting from physiological A50 sol-gel degradation detected in serum at least during the first week. At pathological level, foreign body reaction was present in both of types of catheter, and it was characterized by the presence of macrophages and foreign body giant cell. However, this reaction was different in each group: the A50-coated catheter group showed a higher inflammation with histiocytes, which were forming granuloma-like aggregates with an amorphous crystalline material inside, accompanied by other inflammatory cells such as plasma cells, lymphocytes and mast cells. In conclusion, A50 coating a venous catheter showed excellent bactericidal anti-biofilm response since it completely inhibited S. epidermidis biofilm development and, far from showing procoagulant effects, showed slightly anticoagulant effects.

Toxicity and cross-neutralization of snake venoms from two lesser-known arboreal pit vipers in Southeast Asia: Trimeresurus wiroti and Trimeresurus puniceus

The lesser-known Sundaic lance-headed pit vipers Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia) contribute to the disease burden of snakebite envenomation in Southeast Asia, but their venom toxicity and neutralization remain insufficiently investigated. This study demonstrated that both venoms were procoagulant (involving thrombin-like activity), hemorrhagic, and lethal to mice, with T. wiroti venom being more lethal (LD50 = 0.78 μg/g c.f. 1.21 μg/g). The hetero-specific antivenom from Thailand, Green Pit Viper Antivenom (GPVAV, raised against Trimeresurus albolabris) cross-reacted with T. wiroti and T. puniceus venoms with a higher efficacy of immunological binding activity for the latter. The antivenom was also effective in cross-neutralizing the procoagulant, hemorrhagic and lethal effects of the venoms. In lethality neutralization, GPVAV showed a potency of 0.79–1.05 mg venom per mL antivenom, corresponding to the complete neutralization of approximately 8–10 mg venom per unit vial of antivenom for T. wiroti and T. puniceus venoms. Taken together, it was inferred that T. wiroti, T. puniceus, and T. albolabris venoms share common toxin epitopes, thus enabling the cross-neutralization observed. These findings suggest that GPVAV may be potentially useful in the management of envenomation by T. wiroti and T. puniceus while awaiting clinical trial and validation.

A contradictory action of procoagulant ficin by a fibrinolytic serine protease from Egyptian Ficus carica latex

Ficus carica is one of the most popular and edible plants. Its trees emanate latex of high medical importance. The well-studied procoagulant effect of ficin is a hallmark of this latex which protrudes an interesting question of how can the plant control this effect? In the present work, we purified and characterized a serine protease (FPIII) with fibrinolytic activity from F. carica latex and study the anticoagulant character of the latex. FPIII was inhibited by PMSF and its molecular weight was 48 kDa. The optimum pH and temperature of FPIII were detected at 8.5 and 60 °C, respectively. The activation energy of FPIII was 7 kcal/mol and was thermal stable up to 60 °C. FPIII tended to hydrolyze different protein substrates and showed a good catalytic efficiency (Kcat/Km). The anticoagulant effects and fibrinogenolytic activities of latex crude extract and FPIII were detected, which controls the procoagulant effect of ficin.

LCTX-F2, a Novel Potentiator of Coagulation Factors From the Spider Venom of Lycosa singoriensis.

Spider venoms contain many functional proteins/peptides such as proteinases, serine/cysteine proteinase inhibitors, insecticidal toxins, and ion channel toxins. However, to date, no peptide toxin with procoagulant activities has been identified from spider venom. In this study, a novel toxin LCTX-F2 with coagulation-promoting activity was identified and characterized in the venom of the spider Lycosa singoriensis (L. singoriensis). LCTX-F2 significantly shortened activated partial thromboplastin time (APTT), clotting time, and plasma recalcification time. This toxin directly interacted with several coagulation factors such as FXIIa, kallikrein, thrombin, and FXa and increased their protease activities. In liver bleeding and tail bleeding mouse models, LCTX-F2 significantly decreased the number of blood cells and bleeding time in a dose-dependent manner. At the same dosage, LCTX-F2 exhibited a more significant procoagulant effect than epsilon aminocaproic acid (EACA). Moreover, LCTX-F2 showed no cytotoxic or hemolytic activity against either normal cells or red blood cells. Our results suggested that LCTX-F2 is a potentiator of coagulation factors with the potential for use in the development of procoagulant drugs.