Anticoagulant Protein Research Articles

Global hemostatic profiling in patients with decompensated cirrhosis and bacterial infections

Background & AimsBacterial infections in cirrhosis are associated with increased bleeding risk. To assess the factors responsible for bleeding tendency in patients with bacterial infections, we conducted a prospective study comparing all 3 aspects of hemostasis (platelets, coagulation, and fibrinolysis) in hospitalized patients with decompensated cirrhosis with vs. without bacterial infections.MethodsPrimary hemostasis assessment included whole blood platelet aggregation and von Willebrand factor (VWF). Coagulation assessment included procoagulant factors (fibrinogen, factor II, V, VII, VIII, IX, X, XI, XII, XIII), natural anticoagulants (protein C, protein S, antithrombin) and thrombomodulin-modified thrombin generation test. Fibrinolysis assessment included fibrinolytic factors (plasminogen, t-PA, PAI-1, α2-AP, TAFIa/ai) and plasmin-antiplasmin complex (PAP).ResultsEighty patients with decompensated cirrhosis were included (40 with and 40 without bacterial infections). Severity of cirrhosis and platelet count were comparable between groups. At baseline, patients with cirrhosis and bacterial infections had significantly lower whole blood platelet aggregation, without significant differences in VWF. Regarding coagulation, bacterial infections were associated with reduced procoagulant factors VII and XII, and a significant reduction of all natural anticoagulants. However, thrombomodulin-modified thrombin generation was comparable between the study groups. Finally, although mixed potentially hypo-fibrinolytic (lower plasminogen) and hyper-fibrinolytic (higher t-PA) changes were present in bacterial infections, a comparable level of PAP was detected in both groups. Upon resolution of infection (n = 29/40), platelet aggregation further deteriorated whereas coagulation and fibrinolysis factors returned to levels observed in patients without bacterial infections.ConclusionIn hospitalized patients with decompensated cirrhosis, bacterial infections are associated with reduced whole blood platelet aggregation and a significant decrease of all natural anticoagulants, which may unbalance hemostasis and potentially increase the risk of both bleeding and thrombosis.Lay summaryBacterial infections are a common issue in hospitalized patients with decompensated cirrhosis (i.e. patients hospitalized due to severe complications of advanced chronic liver disease). Patients with decompensated cirrhosis who acquire infections may be at increased risk of bleeding complications following invasive procedures (that is a procedure in which the body is penetrated or entered, for instance by a needle or a tube). As bleeding complications in decompensated cirrhosis are associated with a high risk of further decompensation and death, there is an urgent need to understand the factors responsible for such increased bleeding tendency. Herein, we investigated the alterations of hemostasis (that is the physiological process responsible for clot formation and stability) in patients with decompensated cirrhosis and bacterial infections. We found that development of bacterial infections in these patients is associated with alterations of hemostasis (particularly of platelets and clotting cascade) that may increase the risk of both bleeding and thrombotic complications.

Implications of COVID-19 on Thrombotic Profile of Severely Affected Patients

Introduction: Coronavirus disease 2019 (COVID-19) is a novel viral disease that spread as a global pandemic in 2020 by infecting millions of people across the world. Its clinical prognosis is dependent on various coagulatory parameters since thrombotic events are frequently associated with infection severity. Methods: A total of 383 COVID-19 patients enrolled in Rajiv Gandhi Super Specialty Hospital, Delhi, India, were included in the present retrospective study. Patients were divided into three categories, severe (n = 141), moderate (n = 138), and mild (n = 104) based on infection severity. Various thrombotic parameters and anticoagulant levels were measured in 70 patients and further analyzed. Results: Coagulopathy is seen in COVID-19 patients (n = 70) with a significant increase in fibrinogen, D-dimer levels, and prothrombin time in patients with severe and moderate disease compared to patients with a mild infection. Approximately, 70% of patients with severe and moderate disease demonstrated fibrinogen levels higher than the standard reference range. 60.41% of patients with severe disease showed significantly higher D-dimer levels. Thrombotic parameters were notably elevated in the nonsurvivors group compared to COVID-19 survivors. Nearly, 91% of patients with severe infection had anticoagulant protein S levels below the reference range. Conclusion: COVID-19 infection severely impacts the blood coagulation cascade, which might lead to the manifestation of severe symptoms and increased mortality in patients.

Regulation of the Homeostatic Unfolded Protein Response in Diabetic Nephropathy

A growing body of scientific evidence indicates that protein homeostasis, also designated as proteostasis, is causatively linked to chronic diabetic nephropathy (DN). Experimental studies have demonstrated that the insulin signaling in podocytes maintain the homeostatic unfolded protein response (UPR). Insulin signaling via the insulin receptor non-canonically activates the spliced X-box binding protein-1 (sXBP1), a highly conserved endoplasmic reticulum (ER) transcription factor, which regulates the expression of genes that control proteostasis. Defective insulin signaling in mouse models of diabetes or the genetic disruption of the insulin signaling pathway in podocytes propagates hyperglycemia induced maladaptive UPR and DN. Insulin resistance in podocytes specifically promotes activating transcription factor 6 (ATF6) dependent pathogenic UPR. Akin to insulin, recent studies have identified that the cytoprotective effect of anticoagulant serine protease-activated protein C (aPC) in DN is mediated by sXBP1. In mouse models of DN, treatment with chemical chaperones that improve protein folding provides an additional benefit on top of currently used ACE inhibitors. Understanding the molecular mechanisms that transmute renal cell specific adaptive responses and that deteriorate renal function in diabetes will enable researchers to develop new therapeutic regimens for DN. Within this review, we focus on the current understanding of homeostatic mechanisms by which UPR is regulated in DN.

Purpura Fulminans Triggered by the Formation of Anti-Endothelial Autoantibodies in a Patient with Chronic Lymphocytic Leukemia

Purpura fulminans (PF), a life-threatening disorder characterized by cutaneous microvascular thrombosis with secondary hemorrhagic infarction, is a rare complication of sepsis and may also be caused by severe protein C or protein S deficiency. Here, we describe the case of a 58-year-old man who developed PF in close association with the onset of chronic lymphocytic leukemia (CLL). PF initially manifested with an ecchymosis of the right upper leg in the absence of sepsis or disseminated intravascular coagulation. PF was likely triggered by antiendothelial IgG autoantibodies functionally interfering with the anticoagulant protein C-protein S-thrombomodulin system on EA.hy926 endothelial cells in a modified thrombin generation assay. Although plasma exchange and immunosuppressive therapy with cyclophosphamide were temporarily effective, PF eventually progressed and the patient died from septic shock during treatment-associated neutropenia. In rare occasions, CLL may be associated with autoimmune-mediated PF requiring prompt diagnosis and aggressive multimodal therapy.

Inherited Thrombophilia in the Era of Direct Oral Anticoagulants.

Severe inherited thrombophilia includes rare deficiencies of natural anticoagulants (antithrombin and proteins C and S) and homozygous or combined factor V Leiden and FII G20210A variants. They are associated with a high thrombosis risk and can impact the duration of anticoagulation therapy for patients with a venous thromboembolism (VTE) event. Therefore, it is important to diagnose thrombophilia and to use adapted anticoagulant therapy. The widespread use of direct anticoagulants (DOACs) for VTE has raised new issues concerning inherited thrombophilia. Concerning inherited thrombophilia diagnosis, DOACs are directed toward either FIIa or FXa and can therefore interfere with coagulation assays. This paper reports DOAC interference in several thrombophilia tests, including the assessment of antithrombin, protein S, and protein C activities. Antithrombin activity and clot-based assays used for proteins C and S can be overestimated, with a risk of missing a deficiency. The use of a device to remove DOACs should be considered to minimize the risk of false-negative results. The place of DOACs in the treatment of VTE in thrombophilia patients is also discussed. Available data are encouraging, but given the variability in thrombosis risk within natural anticoagulant deficiencies, evidence in patients with well-characterized thrombophilia would be useful.

Reference interval of antithrombin, protein C, and protein S activities in healthy adults in Iran, the effect of age, sex, oral contraceptive intake, and menopause.

Antithrombin (AT), protein C (PC), and protein S (PS) are natural anticoagulant proteins that deficiency in each of them is associated with an increased risk of venous thromboembolism.The overlapping of plasma levels of AT, PC, and PS between healthy individuals and heterozygote carriers poses significant challenges in precise diagnosis. This study aimed to evaluate the effect of most influencing variables on plasma levels of these proteins and propose specific reference intervals to improve the interpretation of the laboratory results. This study was conducted on 1464 individuals who were referred to Massoud medical laboratory, Tehran, Iran, from 2019 to 2020. AT and PC were measured through chromogenic assay and PS plasma level with the clot-based assay. A multivariable linear regression model was performed to evaluate the effect of sex, age, oral contraceptive (OCP) intake, and menopause state. Normal deviate z value was used for different subgroups to justify the need for a separate reference interval. 1200 verified healthy individuals (434males and 766 females), aged between 18 and 69years were included in the study. The mean±SD age of the participants was 39.78±11.79years. The age-related effects for AT were found in men. In females, increasing age was associated with a rise in AT, PC, and PS plasma levels. No sex difference was found in AT plasma level. OCP-taking is associated with a decrease in AT and an increase in PC plasma levels. This is the largest study ever conducted on healthy individuals in the Iranian population, using specific reference interval results in accurate diagnosis of true AT, PC, and PS deficiency.

Activity of anticoagulant proteins on the polymer-coated and heparin-coated membranes in an extracorporeal circulation circuit.

We performed in vitro experiments using whole human blood without anticoagulants to clarify the activity of anticoagulant proteins on membranes coated with acrylate-copolymer (ACP) with a hydrophilic blood-contacting layer compared to those coated by immobilizing heparin (IHP) in extracorporeal circulation. Whole human blood from healthy volunteers was recirculated in two types of experimental circuits with an ACP-coated reservoir and tubes and an ACP-coated or IHP-coated membrane. To compare the fluctuation of anticoagulant proteins, the circuit pressure at the inlet and outlet of the membrane was measured every 5min; antithrombin antigen (ATQ), antithrombin activity, protein-C quantitation (PCQ), protein-C activity, protein-S free antigen (PSQ), and protein-S activity were measured at 0, 30, 60, 120, and 180min in each experiment (n = 5). The time taken to achieve high circuit pressure (> 300mmHg) at the inlet of the membrane was significantly shorter in the ACP-coated membrane circuit (28 ± 2.7min) than in the IHP-coated membrane circuit (54 ± 24min); however, the ATQ, PCQ, and PSQ at 180min of recirculation were significantly higher in the former than in the latter (all p < .05). ACP-coated membranes can prevent the consumption of anticoagulant proteins but cannot delay circuit thrombogenicity compared to IHP-coated membranes. Considering patient care during the post-extracorporeal circulation period, the use of ACP coating, which can preserve anticoagulant protein, is better in extracorporeal circulation circuits.

Factor VIII/protein C ratio independently predicts liver-related events but does not indicate a hypercoagulable state in ACLD

It has been suggested that the ratio of procoagulant factor VIII to anticoagulant protein C (FVIII/PC) reflects the hemostatic equilibrium. Moreover, FVIII/PC predicted decompensation/death in a small study not accounting for portal hypertension severity. We investigated (i) the prognostic value of FVIII/PC (outcome-cohort) and (ii) whether FVIII/PC reflects the hypercoagulable state (assessed by thrombomodulin-modified thrombin generation assay [TM-TGA]) or the risk of bleeding/thrombotic events in patients undergoing hepatic venous pressure gradient (HVPG) measurement during follow-up. (i) The outcome-cohort comprised 576 patients with evidence of advanced chronic liver disease (liver stiffness measurement ≥10 kPa and/or HVPG ≥6 mmHg). (ii) TM-TGA-cohort patients (n= 142) were recruited from the prospective VIenna CIrrhosis Study (VICIS: NCT03267615). (i) FVIII/PC significantly increased across clinical stages (p <0.001) as well as HVPG (p <0.001) and MELD score (p <0.001) strata and remained independently associated with decompensation/liver-related death (adjusted hazard ratio 1.06; 95% CI 1.01-1.11; p= 0.013), even after multivariable adjustment. It was also associated with acute-on-chronic liver failure (ACLF) development (adjusted hazard ratio 1.10; 95% CI 1.02-1.19; p= 0.015) in patients with decompensated cirrhosis. (ii) FVIII/PC showed a weak positive correlation with endogenous thrombin potential (Spearman's ρ= 0.255; p= 0.002), but this association disappeared after adjusting for the severity of liver disease. FVIII/PC was not associated with the development of bleeding (p= 0.272) or thrombotic events (p= 0.269). However, FVIII/PC correlated with biomarkers of different pathophysiological mechanisms that promote liver disease progression. FVIII/PC provides prognostic information regarding hepatic decompensation/death and ACLF, independently of established prognostic indicators. However, this is not evidence that hypercoagulability drives disease progression, as the correlation between FVIII/PC and thrombin generation is confounded by liver disease severity and FVIII/PC was not associated with thrombosis. Therefore, FVIII/PC does not reflect coagulation and results from previous studies on FVIII/PC require re-interpretation. NCT03267615 (in part). A balanced coagulation system is essential for preventing bleeding episodes and blood clot formation (thrombosis). Blood of patients with advanced liver disease may have increased coagulation potential, possibly promoting the worsening of liver disease via thrombosis in the blood vessels of the liver. The ratio between the results of 2 blood tests (procoagulant factor VIII to anticoagulant protein C) has been suggested to reflect these increases in coagulation potential. Our study demonstrates, on the one hand, that this ratio is a versatile predictor of the development of complications of cirrhosis, yet on the other hand, that it is unrelated to coagulation.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman

Ethnopharmacological relevanceThe prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. Aim of the studyWe aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. Materials and methodsW. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. ResultsThe anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2–8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. ConclusionOur results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

Rationale and design of a study to assess the safety and efficacy of rNAPc2 in COVID-19: the Phase 2b ASPEN-COVID-19 trial

BackgroundThe interaction between thrombosis and inflammation appears central to COVID-19-associated coagulopathy and likely contributes to poor outcomes. Tissue factor is a driver of disordered coagulation and inflammatory signaling in viral infections and is important for viral replication; therefore, tissue factor may be an important therapeutic target in COVID-19.Study DesignASPEN-COVID-19 (NCT04655586) is a randomized, prospective open-label blinded endpoint (PROBE), active comparator Phase 2b trial to evaluate the safety and efficacy of recombinant Nematode Anticoagulant Protein c2 (rNAPc2), a potent tissue factor inhibitor, in patients hospitalized with COVID-19 with elevated D-dimer levels. This report describes the design of the Phase 2b dose ranging and proof of concept study. Participants are randomly assigned, in a 1:1:2 ratio, to lower or higher dose rNAPc2 by subcutaneous injection on days 1, 3, and 5 or to heparin according to local standard of care; randomization is stratified by baseline D-dimer level (at 2X upper limit of normal). The primary efficacy endpoint for Phase 2b is proportional change in D-dimer concentration from baseline to Day 8 or day of discharge, whichever is earlier. The primary safety endpoint is major or non-major clinically relevant bleeding through Day 8. Phase 2b enrollment began in December 2020 and is projected to complete ∼160 participants by Q4 2021.ConclusionsASPEN-COVID-19 will provide important data on a novel therapeutic approach that may improve outcomes in hospitalized COVID-19 patients beyond available anticoagulants by targeting tissue factor, with potential effects on not only thrombosis but also inflammation and viral propagation.

Significance of altered anticoagulant proteins and D-dimer in cirrhotic portal vein thrombosis: relation to the degree of liver dysfunction.

Portal vein thrombosis (PVT) is a well-known consequence of cirrhosis. Its pathophysiology is complex, with possible downstream hepatic decompensation. This study was conducted to describe the changes of protein C (PC), protein S (PS) and D-dimer blood levels associated with PVT formation in cirrhosis and the relation to the degree of liver dysfunction. This was a case-control study that included 50 cirrhotic patients who presented with acute de novo non-malignant PVT and 50 cirrhotic patients without PVT as a control group. The severity of liver disease was classified as per the Child-Turcotte-Pugh (CTP) score. Doppler ultrasonography identified acute portal vein occlusion, and dynamic contrast-enhanced computed tomography confirmed the extent and nature of PVT. Blood PC, PS and D-dimer levels were measured using enzyme-linked immunosorbent assay. PC and PS levels were significantly lower, and the D-dimer level was significantly higher, in cirrhotic patients with PVT compared to the control group. PC and PS levels were significantly decreased in patients with higher CTP score of both groups. The D-dimer level did not vary significantly with the degree of liver dysfunction in patients of either group. PC, PS and D-dimer at the cut-off points of ≤ 77 IU/dl, ≤ 63 IU/dl, and > 300 ng/ml, respectively, significantly suggested PVT occurrence. Alteration of the anticoagulant proteins and D-dimer contributed to PVT formation in cirrhotic patients and could help stratify the degree of liver dysfunction. Blood level of these hemostatic proteins could be incorporated into a probability score for early diagnosis and treatment of PVT in cirrhosis.

Clinical guidance for the peripartum management of patients with hereditary thrombophilia

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in the anticoagulant factors antithrombin (AT), protein C, or protein S. The recommendations for peripartum management are as follows: 1) For women with acute venous thromboembolism (VTE) during pregnancy, anticoagulant therapy with a therapeutic dose of unfractionated heparin (UFH) is recommended; 2) For women with a history of VTE, a prophylactic dose of UFH is suggested during pregnancy; 3) For those with AT deficiency, supplementation with an AT preparation in addition to UFH is suggested; 4) For women with no history of VTE, anticoagulant therapy during pregnancy is considered for each thrombophilia type; 5) When anticoagulant therapy consisting of a prophylactic dose of UFH is administered during pregnancy, injection is discontinued with the onset of labor pains in cases of vaginal delivery and 6 hours before the start of delivery in cases of planned delivery or cesarean section; 6) In cases of AT deficiency, regardless of the thrombophilia type, supplementation with AT before and after delivery is suggested; 7) For women with thrombophilia and a history of VTE and for those who receive anticoagulant therapy during pregnancy, postpartum anticoagulant therapy is recommended.

Protein S: a Central Regulator of Blood Coagulation.

Protein S is a central regulator of coagulation as it critically participates in down-regulation of both extrinsic and intrinsic pathways of the coagulation cascade. In this review, we aim to provide an update on protein S and its anticoagulant functions as a central hemostatic regulator. Electronic databases including, Google, Google Scholar, PMC, PubMed, Science Direct, and Scopus were rigorously searched using the terms protein S, hemostasis, natural anticoagulants, regulators of coagulation, and coagulation inhibitors for the completion of this descriptive review. Literature review shows that protein S is a potent cofactor for activated protein C (APC) in the regulation of the intrinsic pathway and a cofactor for tissue factor pathway inhibitor (TFPI) in the regulation of the extrinsic pathway. The strong association between protein S deficiency either hereditary or acquired and increased risk for venous thrombosis indicates the important and central role of protein S in controlling the initiation and propagation phase of coagulation cascade and that protein S is an important determinant for optimal activity of both APC and TFPI in coagulation regulation. Available evidence suggests that the role of protein S in the down-regulation of blood coagulation is mainly mediated through its high affinity binding to negatively charged phospholipid surfaces. This high affinity binding to negatively charged phospholipids helps bring the anticoagulant proteins to the membranes, resulting in efficient and targeted regulation of coagulation. In the shade of current COVID-19 pandemic, protein S deficiency has been found to be a leading cause of thrombotic complications associated with COVID-19.

Kallikrein augments the anticoagulant function of the protein C system in thrombin generation

BackgroundGenetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)‐modulated TG using plasma from the Human Functional Genomics Project. MethodsCalibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome‐wide single‐nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A‐IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. ResultsThrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome‐wide level (nETP‐TMsr, P = 4.27 × 10−8). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A‐IV, into plasma dose‐dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP‐TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor. ConclusionsOur results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis.

Purpura Fulminans in a 20-Year-Old Female

&#x0D; &#x0D; &#x0D; A previously healthy 20-year-old female presented with extensive retiform purpura located at the face, upper and lower limbs, one week after an episode of acute tonsillitis. Despite the exuberance of the cutaneous findings and progression to skin necrosis she had no accompanying symptoms. Laboratory investigation revealed a heterozygous protein C mutation (exon 9, c.1332G&gt; C, p.Trp444Cys), accounting for a partial deficiency of this anticoagulant protein. The patient was started on broad spectrum antibiotics, anticoagulation and systemic corticosteroids, with no lesional progression and complete resolution of cutaneous ulceration within 6 months.&#x0D; This is a singular case of purpura fulminans, since two different causative factors precipitated the events. The previous tonsillitis reported by the patient is significant, because the serum concentration of protein S may also decrease after an infectious event - post-infectious purpura fulminans. This case illustrates that purpura fulminans due to autoantibodies against protein S, although rare, should be considered, especially in the absence of a severe acute infection. It also illustrates how in a given patient different independent factors can act simultaneously, triggering potentially devastating clinical scenarios.&#x0D; &#x0D; &#x0D;

Molecular mechanisms of hemostasis impairment in oncology

Malignant neoplasms are characterized by the presence of the hemostasis system pathology, predisposing cancer patients to thrombohemorrhagic complications. The pathogenesis of cancer-associated coagulopathy is complex and involves a variety of mechanisms. Tumor cells have the ability to activate the host’s hemostasis system, and this phenomenon is controlled by the same oncogenes that are responsible for neoplastic transformation. In addition to predisposing factors to impaired hemostasis from the side of the disease, the anticancer drugs themselves carry risks of developing coagulation disorders. The pathophysiological basis of this kind of disorders caused by chemotherapy is associated with damage to the endothelium, imbalance of coagulation and anticoagulant proteins, platelet dysfunction and their deficiency. In this article, the authors set themselves the goal of generalizing and updating the current knowledge of the molecular mechanisms that cause thrombohemorrhagic risk in cancer.

Cardiac inflammation and microvascular procoagulant changes are decreased in second wave compared to first wave deceased COVID-19 patients

BackgroundCompelling evidence has shown cardiac involvement in COVID-19 patients. However, the overall majority of these studies use data obtained during the first wave of the pandemic, while recently differences have been reported in disease course and mortality between first- and second wave COVID-19 patients. The aim of this study was to analyze and compare cardiac pathology between first- and second wave COVID-19 patients. MethodsAutopsied hearts from first- (n = 15) and second wave (n = 10) COVID-19 patients and from 18 non-COVID-19 control patients were (immuno)histochemically analyzed. CD45+ leukocyte, CD68+ macrophage and CD3+ T lymphocyte infiltration, cardiomyocyte necrosis and microvascular thrombosis were quantified. In addition, the procoagulant factors Tissue Factor (TF), Factor VII (FVII), Factor XII (FXII), the anticoagulant protein Dipeptidyl Peptidase 4 (DPP4) and the advanced glycation end-product N(ε)-Carboxymethyllysine (CML), as markers of microvascular thrombogenicity and dysfunction, were quantified. ResultsCardiac inflammation was significantly decreased in second wave compared to first wave COVID-19 patients, predominantly related to a decrease in infiltrated lymphocytes and the occurrence of lymphocytic myocarditis. This was accompanied by significant decreases in cardiomyocyte injury and microvascular thrombosis. Moreover, microvascular deposits of FVII and CML were significantly lower in second wave compared to first wave COVID-19 patients. ConclusionsThese results show that in our cohort of fatal COVID-19 cases cardiac inflammation, cardiomyocyte injury and microvascular thrombogenicity were markedly decreased in second wave compared to first wave patients. This may reflect advances in COVID-19 treatment related to an increased use of steroids in the second COVID-19 wave.

Protein S-mediated signal transduction pathway regulates lung cancer cell proliferation, migration and angiogenesis

Objective/BackgroundProtein S (PS; encoded by the PROS1 gene), a key vitamin K-dependent anticoagulant protein, is emerging as a key structural and functional protein that is overexpressd in various malignancies, but how PS signals to promote lung cancer progression is unclear. MethodsWe used immortalized, nontumorigenic human lung epithelial cell line NL-20, A549 cells as experimental cellular models for lung cancer, and human microvascular endothelial cells (HMEC-1) as a model system for angiogenesis. A loss- and gain-of-function approach was then used to analyze the role of tumor-derived PS and their natural TAM receptors Tyro3 and MerTK in regulating cell proliferation, migration, anchorage-independent growth, and capillary-like tube formation, all prominent attributes of the metastatic phenotype of tumor cells. ResultsEvidence is now provided that regulation of PROS1 gene expression using either stable cell lines expressing lentiviral-short hairpin RNA (shRNAs) or a replication-incompetent adenovirus alters the phosphorylation of several major signaling pathways, including Erk, PKB/Akt, p38, and focal adhesion kinase (FAK), and modulates PS-dependent Tyro3- and MerTK-mediated cell migration, proliferation, and anchorage-independent growth of lung cancer cells, and endothelial cell capillary-like tube formation. ConclusionThese finding suggest that the PS-Tyro3 and -MerTK axis mediates important signaling pathways to promote lung cancer progression. Genetic inhibition of endogenous PS may serve as a promising target for anticancer drug development.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host’s blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

Constitutive Expression of Hypoxia Inducible Factor Downregulates the Anticoagulant Protein S in a Unique Chuvash Polycythemia Population with Erythrocytosis

Background: Chuvash polycythemia is a hematological disorder present worldwide but endemic to the Chuvash population, a Turkish ethnic group in Russia. The disorder is caused by a homozygous germline mutation (R200W) in the von Hippel Lindau gene (1,2). This mutation impairs binding of pVHL to hypoxia-inducible factor 1-alpha (HIF-1a); lack of this interaction prevents degradation of HIF-1a (2,3). The resultant upregulation of HIF-1a, even in a normal oxygen state, increases the activity of erythropoietin, thereby causing polycythemia (2,3). Affected individuals experience increased rates of arterial and venous thrombosis unrelated to the increased concentration of hemoglobin (4,5).Aims: To determine whether upregulation of HIF-1a in Chuvash polycythemia individuals causes a decreased level of the antithrombotic Protein S. A decreased level of Protein S may explain the increased risks of thrombotic events and vascular abnormalities in the Chuvash population.Methods: Enzyme-linked immunosorbent assays (ELISA) were performed to measure total Protein S concentration in Chuvash and control plasma. Immunoblotting was performed to confirm the ELISA measurements. Additional assays to measure free Protein S and thrombin generation assays with and without Protein S will be performed to determine whether Chuvash plasma has low free Protein S and whether Protein S supplementation will improve the thrombotic phenotypes.Results: Total Protein S concentration measured by ELISA was lower in Chuvash individuals compared with controls. In addition, we have completed immunoblotting of seven Chuvash samples and three control samples. Our results indicated that Chuvash plasma had lower amounts of Protein S compared with the controls. Immunoblotting of additional Chuvash samples is in progress, and we are planning to measure the free Protein S in Chuvash samples.Conclusion: Our preliminary results suggest a decreased level of total Protein S in individuals with Chuvash polycythemia. Because an increased hemoglobin concentration does not increase the rates of thromboembolic events in this population, our finding of a reduced amount of anticoagulant Protein S may explain the hypercoagulability that Chuvash individuals experience.