Thrombin Activity Research Articles

The Oligosaccharide of Human Thrombin: Investigations of Functional Significance

Functional properties of the carbohydrate chain of human thrombin were examined by quantitating the activity of the enzyme before and after partial removal of its oligosaccharide by exoglycosidases. The following activities were studied: fibrinogen clotting, factor VIII coagulant (VIII:C) activation, inhibition by defibrinated plasma (anti-thrombin-III and alpha 2-macroglobulin), binding to polymerized fibrin, and stimulation of platelet release and aggregation. In general, the published information about the activity of native thrombin in these interactions was confirmed, though differences were observed in the association constants describing thrombin binding to fibrin. Partial deglycosylation had no apparent effect on any of these activities. It is concluded, therefore, that the oligosaccharide of human thrombin is located outside the major protein and platelet-binding regions of the molecule.

Effect of Active Site-Modified Thrombin on the Hydrolysis of Platelet-Associated Glycoprotein V by Native Thrombin

To determine the relationship between equilibrium binding of thrombin to sites on the platelet surface and the cleavage of membrane glycoprotein V (GPV) by thrombin, we examined the effect of active site-modified thrombin (1-chloro-3-tosylamido-7-amino-L-2-heptanone thrombin toslysCH2-thrombin) on the binding of native thrombin to platelets and on the hydrolysis of GPV by native thrombin. ToslysCH2-thrombin inhibited binding of native thrombin to high affinity sites on the platelet surface. In contrast, hydrolysis of GPV by native thrombin, even at threshold thrombin concentrations, was not inhibited by pretreatment with toslysCH2-thrombin at concentrations up to 210 nmol/L. ToslysCH2-thrombin also had no appreciable effect on platelet aggregation or release of 14C-serotonin induced by native thrombin. Because toslysCH2-thrombin does not inhibit platelet release, aggregation, or GPV hydrolysis by native thrombin but does inhibit high affinity surface binding by native thrombin, these results indicate that thrombin binding and hydrolysis of GPV are separate and unrelated events.

Hirudin prevents vascular endothelial cell apoptosis and permeability enhancement induced by the serum from rat with chronic renal failure through inhibiting RhoA/ROCK signaling pathway.

Endothelial cells injury and activation contribute to arteriovenous fistula (AVF) stenosis. Hirudin (Hiru) can inhibit the activity of thrombin, which was reported to enhance endothelial cell permeability and promote vascular inflammatory responses. RhoA/ROCK signaling pathway is also important in regulating vascular endothelial permeability. This study aimed to investigate the role of Hiru on the viability and permeability of human umbilical vein endothelial cells (HUVECs) following stimulation of serum from rat with chronic renal failure (CRF) and illustrated the effects of Hiru on RhoA/ROCK signaling. Wistar rats were randomly divided into control group and CRF group. Serum from each group was collected to stimulate HUVECs. Proliferation capability was estimated with Cell Count Kit-8 (CCK-8) assay. Transwell assay was performed to determine permeability. Cell apoptosis was examined using Tunel staining. Telomere length and telomerase activity were determined by qPCR. Moreover, the expression of RhoA, ROCK1 and ROCK2 was estimated via western blot. Results showed that the serum from CRF rat significantly inhibited cell viability while enhanced cell permeability and apoptosis. Different concentrations of Hiru prevented the above effects caused by CRF serum. Additionally, Hiru recovered the CRF serum-induced decreased telomere length and telomerase activity. Hiru also inhibited the protein expression of RhoA, ROCK1 and ROCK2, which were activated by CRF serum. Moreover, the ROCK inhibitor, Y27632, exhibited similar effects with Hiru. In conclusion, Hiru-restored HUVECs cell viability, telomere length and telomerase activity, suppressed permeability and apoptosis in the presence of CRF serum might depend on inactivating the RhoA/ROCK signaling.

Chromogenic anti-Xa test: the ratio between heparin activity units and concentration of apixaban and rivaroxaban

Introduction. The direct oral anticoagulants (DOC) therapy does not require alaboratory control; however, it may be required to determine the anticoagulationlevel to choose a treatment strategy if alarge bleeding is developing or emergency surgery is needed.The objective of this experimental study was to investigate the relationship between the residual factor Xa (FXa) activity, anti-Xa activity units oflow molecular weight heparins (LMWH), and the apixaban and rivaroxaban plasma concentrations in a chromogenic anti-Xa assay.Material and methods. Concentrated DOC solutions were prepared by extracting apixaban and rivaroxaban from crushed tablets using methanol and dimethyl sulfoxide, respectively. The resulting solutions were added to the donor plasma pool until final inhibitor concentrations are achieved in the range from 10 to 100 ng/ml plasma. Anti-Xa activity was determined using an STA-compact analyser and the Liquid anti-Xa reagent kit, an analysis protocol, and calibrators designed to control the LMWH therapy. The effect on the thrombin formation dynamics was investigated using the thrombin generation test (TGT) and the PPR reagent as a trigger (final concentrations of tissue factor are 5 pM, and those of phospholipids are 4 μM). TGT curves were analysed using the Thrombinoscope program.Results. It was shown that in the anti-Xa activity test version designed to control the LMWH therapy, there is a high correlation (R2 > 0.98) between thelogarithm of the residual factor Xa activity and the content of apixaban and rivaroxaban in the range from 10 to 80 ng/ml. Rivaroxaban shows about 1.5 times more anti-Xa activity than apixaban at equal concentrations. It was also shown that apixaban and rivaroxaban at doses equal both in concentration and in anti-Xa activity differ in their effect on the thrombin formation dynamics and thrombin inactivation in the TGT.Conclusion. In the LMWH anti-Xa activity test version, the measured range of apixaban and rivaroxaban includes 30 ng/ml and 50 ng/ ml concentrations taken as “cut-off points” to determine the treatment tactics in emergency cases. However, thelack of certified DOC calibratorslimits the use of this test in clinical practice.

Changes in the Coagulation Mechanism Following Wounding and Resuscitation with Stored Blood: A Study of Battle Casualties in Korea

1. The coagulation system was studied in eleven battle casualties immediately after they had been wounded and resuscitated and during the first days of their convalescence. 2. In general the clotting time was shorter than normal and the platelet count and fibrinogen concentration were greater than normal. Prothrombin activity as measured by the one-stage test averaged 50 per cent of normal immediately after resuscitation with large transfusions of stored blood. This defect corrected itself within one to three days, but about the fourth day prothrombin activity again was reduced to about 50 per cent of normal and thereafter recovered gradually. 3. The primary fall of prothrombin activity was not proved to be caused by a lack of prothrombin but appeared in some cases to be related to a lack of labile factor which may have been a consequence of large transfusions of stored blood deficient in the labile factor. The prothrombin time was not always corrected by addition of labile factor. The defect of the coagulation mechanism was never great enough to cause serious hemorrhage. A tendency to ooze was noted in patients who had received over 20 pints of blood in a short time. It is suggested that this may have been due to the presence of a great number of immature platelets in the circulation. No fibrinolytic reactions were observed. 4. The secondary fall of prothrombin activity was not due to the lack of prothrombin. The defect of the coagulation mechanism could be corrected by the addition of heated serum that was free of prothrombin, thrombin, labile factor, stable factor, and thromboplastic activity. The factor responsible for the correction of the coagulation defect was heat stable (56 C. for 30 minutes in the presence of oxalate) and was not absorbed by tricalcium phosphate gel. Of the known factors involved in the coagulation system, these two characteristics eliminate all except the platelet factor 3. This fall in prothrombin activity was not associated with any clinical hemorrhagic tendency.

Human Neutrophil Elastase Alters Human α-Thrombin Function: Limited Proteolysis Near the γ-Cleavage Site Results in Decreased Fibrinogen Clotting and Platelet-Stimulatory Activity

During blood coagulation, polymorphonuclear leukocytes release elastase in amounts that can exceed 100 nmol/L. We therefore studied the interaction between human leukocyte elastase and human alpha-thrombin. Elastase cleaved the thrombin B chain (Ala 150-Asn 151) near the gamma-cleavage site, resulting in two fragments held together by noncovalent interactions. The NH2-terminal fragment (FI), mol wt approximately 18,000, was disulfide-linked to the thrombin A chain. The COOH-terminal fragment (FII), mol wt approximately 13,000, contained the active-site serine and formed a covalent bond with antithrombin III. Heparin accelerated proteolysis of alpha-thrombin by elastase. Proteolyzed alpha-thrombin (T theta) retained full amidolytic activity; however, the concentration of T theta causing 50% maximal platelet aggregation and adenosine triphosphate (ATP) release was 7.9 nmol/L (1.1 nmol/L for alpha-thrombin and 220 nmol/L for gamma-thrombin). Fibrinogen clotting activity of T theta and gamma-thrombin was 32% and 1% that of alpha-thrombin, respectively. Elastase released during the coagulation process may modulate thrombin activity. In addition, elastase-modified thrombin may be a useful probe of the structure and function of the gamma-cleavage region.

Protease-activated receptor 1 as a potential therapeutic target for COVID-19.

Acute respiratory disease caused by a novel coronavirus (SARS-CoV-2) has spread all over the world, since its discovery in 2019, Wuhan, China. This disease is called COVID-19 and already killed over 1 million people worldwide. The clinical symptoms include fever, dry cough, dyspnea, headache, dizziness, generalized weakness, vomiting, and diarrhea. Unfortunately, so far, there is no validated vaccine, and its management consists mainly of supportive care. Venous thrombosis and pulmonary embolism are highly prevalent in patients suffering from severe COVID-19. In fact, a prothrombotic state seems to be present in most fatal cases of the disease. SARS-CoV-2 leads to the production of proinflammatory cytokines, causing immune-mediated tissue damage, disruption of the endothelial barrier, and uncontrolled thrombogenesis. Thrombin is the key regulator of coagulation and fibrin formation. In severe COVID-19, a dysfunctional of physiological anticoagulant mechanisms leads to a progressive increase of thrombin activity, which is associated with acute respiratory distress syndrome development and a poor prognosis. Protease-activated receptor type 1 (PAR1) is the main thrombin receptor and may represent an essential link between coagulation and inflammation in the pathophysiology of COVID-19. In this review, we discuss the potential role of PAR1 inhibition and regulation in COVID-19 treatment.

Activated Factor XI is Increased in Plasma in Response to Surgical Trauma but not to Recombinant Activated FVII-Induced Thrombin Formation.

Aim: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. Methods: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n =89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE−), and patients undergoing major orthopedic surgeries ( n =45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin–antithrombin complex (TAT). Results: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively ( P =4·10 −4 ) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L ( P =0.001). Peak levels of F1+2 were comparable in the VTE+, VTE−, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). Conclusions: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

Magnolol, A Novel Antagonist of Thrombin and PAR-1, Inhibits Thrombin-Induced Connective Tissue Growth Factor (CTGF) Expression in Vascular Smooth Muscle Cells and Ameliorate Pathogenesis of Restenosis in Rats

Restenosis and destructive vascular remodeling are the main reasons for treatment failure in patients undergoing percutaneous coronary intervention (PCI). In this study, we explored the efficacy of magnolol (a traditional Chinese medicine) in the treatment of restenosis. The results of this study showed that the activities of thrombin and PAR-1 (protease-activated receptor 1) were significantly decreased by the treatment of magnolol. Based on protein docking analysis, magnolol exhibits its potential to bind to the PAR-1 active site. In addition, thrombin-induced connective tissue growth factor (CTGF) expression and the upstream of CTGF such as JNK-1 (but not JNK-2), c-Jun, and AP-1 were distinctly inhibited by magnolol (50 μM) in vascular smooth muscle cells (VSMC). For the functional assay, magnolol (50 μM) significantly inhibited the migration of VSMC, and rats treated with magnolol (13 mg/kg/day) after balloon angioplasty has observed a significant reduction in the formation of common arterial neointima. In conclusion, we identified a novel mechanism by which magnolol acts as the thrombin activity inhibitor and may be the PAR-1 antagonist. In accordance with these functions, magnolol could decrease thrombin-induced CTGF expression in VSMCs via PAR-1/JNK-1/AP-1 signaling.

Accuracy of Fibrinogen/D-dimer Ratio in Predicting the Occurrence of Coronary Slow Flow Phenomenon

BACKGROUND: Fibrinogen is a risk factor for coronary heart disease (CHD), which is an acute-phase protein that is released when inflammation occurs. Fibrinogen is an essential component in the coagulation cascade and affects hemostasis, hemorheology, platelet aggregation, and endothelial function. D-dimers are inevitable products of fibrin degradation that results from thrombin activation; XIII activated factors and plasmin.
 AIM: This study was aimed to explore the accuracy of the fibrinogen/D-dimer ratio in predicting the slow flow phenomenon in CHD.
 METHODS: The study design is a diagnostic test with a cross-sectional design to assess the accuracy of the fibrinogen/d-dimer ratio in predicting coronary slow flow phenomenon (CSFP) in CHD. The study was conducted at the Department of Internal Medicine, Faculty of Medicine, Dr. Moh Hoesin Hospital, Palembang, Indonesia. The independent variables in this study were serum fibrinogen and D-dimer levels. Meanwhile, the dependent variable is sufferers of CHD with CSFP and regular flow. All data obtained from this study were analyzed using the SPSS version 20.0 for Windows program with a 95% confidence limit.
 RESULTS: This study showed that there was no difference in levels of fibrinogen and D-dimer in groups with CSFP and regular flow. Fibrinogen and D-dimer do not play a significant role in the CSFP process. The various etiological parts of this possible disorder still need further exploration and detail, ranging from the role of the renin-angiotensin-aldosterone system, the function of the autonomic nervous system, to the position of other endothelial factors.
 CONCLUSION: This study proves that fibrinogen has specific but not sensitive predictors of CSFP. The d-dimer value is also not accurate as a CSFP predictor. This study demonstrates that the fibrinogen / d-dimer ratio is not precise as a predictor of CSFP events.

Factor XI, much more than an innocent observer

Factor XI, much more than an innocent observer

Pharmacodynamics of eftrenonacog-alfa (rFIX-Fc) in severe hemophilia B patients: A real-life study

Eftrenonacog-alfa is a recombinant factor IX-Fc fusion protein increasingly prescribed in hemophilia B patients. We aimed to assess its pharmacodynamics (PD) in real-life setting via FIX activity measurement and thrombin generation assay (TGA).Sixty samples from 15 severe hemophilia B treated patients were collected at different time points. FIX activity was measured using product-specific one-stage clotting assay (reference method) and two chromogenic assays (CSA) (Biophen FIX and Rox FIX). TGA was triggered with 1 pM tissue factor. Five parameters were analyzed: lag time (LT), time to peak (TTP), peak height (PH), endogenous thrombin potential (ETP), and velocity. PD models were built to characterize their relationships with FIX activity, using mixed effects models.Mean trough FIX level was estimated at 4.64 (±1.50) IU/dl with a recovery at 0.78 (±0.16) IU/dl per 1 IU/kg injected dose. FIX activity ranged between 1 and 86 IU/dl with 21.5 IU/dl median value. Biophen FIX and Rox FIX allowed reliable measurements except in samples with FIX <20 IU/dl in which values were underestimated (delta >30%). PD models revealed that velocity was the most sensitive TGA parameter to FIX activity followed by PH, ETP, TTP and finally LT. Following FIX activity peak after eftrenonacog-alfa injection, velocity decreased first, followed by PH then ETP.Both CSA failed to accurately measure FIX in severe hemophilia B patients receiving eftrenonacog-alfa throughout the measuring range. TGA could be an additional valuable tool to evaluate hemostasis balance in treated patients.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

A novel serine protease with anticoagulant and fibrinolytic activities from the fruiting bodies of mushroom Agrocybe aegerita

A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, β and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway.

Increased Mucosal Thrombin is Associated with Crohn's Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation.

Background and AimsThrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn’s disease.MethodsThrombin activity was studied in tissues from Crohn’s disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid.ResultsActive forms of thrombin were increased in Crohn’s disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models.ConclusionsOur data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

High CSF thrombin concentration and activity is associated with an unfavorable outcome in patients with intracerebral hemorrhage.

The cerebral thrombin system is activated in the early stage after intracerebral hemorrhage (ICH). Expression of thrombin leads to concentration dependent secondary neuronal damage and detrimental neurological outcome. In this study we aimed to investigate the impact of thrombin concentration and activity in the cerebrospinal fluid (CSF) of patients with ICH on clinical outcome. Patients presenting with space-occupying lobar supratentorial hemorrhage requiring extra-ventricular drainage (EVD) were included in our study. The CSF levels of thrombin, its precursor prothrombin and the Thrombin-Antithrombin complex (TAT) were measured using enzyme linked immune sorbent assays (ELISA). The oxidative stress marker Superoxide dismutase (SOD) was assessed in CSF. Initial clot size and intraventricular hemorrhage (IVH) volume was calculated based on by computerized tomography (CT) upon admission to our hospital. Demographic data, clinical status at admission and neurological outcome were assessed using the modified Rankin Scale (mRS) at 6-weeks and 6-month after ICH. Twenty-two consecutive patients (9 females, 11 males) with supratentorial hemorrhage were included in this study. CSF concentrations of prothrombin (p < 0.005), thrombin (p = 0.005) and TAT (p = 0.046) were statistical significantly different in patients with ICH compared to non-hemorrhagic CSF samples. CSF concentrations of thrombin 24h after ICH correlated with the mRS index after 6 weeks (r2 = 0.73; < 0.005) and 6 months (r2 = 0.63; < 0.005) after discharge from hospital. Thrombin activity, measured via TAT as surrogate parameter of coagulation, likewise correlated with the mRS at 6 weeks (r2 = 0.54; < 0.01) and 6 months (r2 = 0.66; < 0.04). High thrombin concentrations coincide with higher SOD levels 24h after ICH (p = 0.01). In this study we found that initial thrombin concentration and activity in CSF of ICH patients did not correlate with ICH and IVH volume but are associated with a poorer functional neurological outcome. These findings support mounting evidence of the role of thrombin as a contributor to secondary injury formation after ICH.

The Research Progress of Direct Thrombin Inhibitors.

Blood coagulation is the process of changing the blood from the flowing state to the gel state. It is an important part of the hemostatic function. Coagulation is a process by which a series of coagulation factors are sequentially activated, and finally thrombin is formed to form fibrin clot. Direct thrombin inhibitors are important anticoagulant drug. These drugs can selectively bind to the active site of thrombin, inhibit thrombin activity, have strong action and high specificity, and have important significance in the clinical treatment of thrombus diseases. Some of them come from natural products of animals or plants, and many of them have been applied in the clinic. The other part is derived from the design, synthesis and activity studies of small molecule inhibitors. This review discusses the progress of direct thrombin inhibitors in recent years.

Tamoxifen induces hypercoagulation and alterations in ER\u03b1 and ER\u03b2 dependent on breast cancer sub-phenotype ex vivo

Tamoxifen shows efficacy in reducing breast cancer-related mortality but clinically, is associated with increased risk for thromboembolic events. We aimed to determine whether breast tumour sub-phenotype could predict propensity for thrombosis. We present two ex vivo Models of Tamoxifen-therapy, Model 1 in which treatment recapitulates accumulation within breast tissue, by treating MCF7 and T47D cells directly prior to exposure to blood constituents; and Model 2 in which we recreate circulating Tamoxifen by treating blood constituents prior to exposure to cancer cells. Blood constituents included whole blood, platelet-rich plasma and platelet-poor plasma. Hypercoagulation was assessed as a function of thrombin activity, expression of CD62P and CD63 activation markers defined as an index of platelet activation, and platelet morphology; while oestrogen receptor expression was assessed using immunocytochemistry with quantitative analysis. We determined, in concert with clinical studies and contrary to selected laboratory investigations, that Tamoxifen induces hypercoagulation, dependent on sub-phenotypes, with the T47D cell line capacity most enhanced. We determined a weak positive correlation between oestrogen receptor expression, and CD62P and CD63; indicating an association between tumour invasion profiles and hypercoagulation, however, other yet unknown factors may play a predictive role in defining hypercoagulation.

Preclinical Characterization of BCX9930, a Potent Oral Complement Factor D Inhibitor, Targeting Alternative Pathway-Mediated Diseases Including Paroxysmal Nocturnal Hemoglobinuria (PNH)

INTRODUCTION: Complement factor D is a serine protease that specifically catalyzes the cleavage of factor B [bound to C3(H2O) or C3b], its only natural substrate, to Ba and Bb. Factor D has the lowest concentration in plasma among all the complement proteins and is the rate-limiting enzyme of the alternative pathway (AP) of complement. Factor D plays a key role in both AP-initiated C3 convertase formation and the amplification loop of the complement cascade. Therefore, targeting factor D is a promising therapeutic strategy to inhibit AP activation for treatment of AP-mediated diseases such as PNH, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Inhibition of terminal complement C5 by eculizumab or ravulizumab results in sustained inhibition of intravascular hemolysis in patients with PNH by blocking formation of the membrane attack complex (C5b-9). However, many PNH patients treated with eculizumab continue to have symptomatic anemia, with up to half of the patients requiring blood transfusions due to extravascular hemolysis as a result of ongoing AP activation and C3 opsonization on erythrocytes. BioCryst Pharmaceuticals has developed a small-molecule, orally bioavailable human factor D inhibitor that is currently under evaluation as oral monotherapy for the treatment of patients with PNH in a Phase 1 study (BCX9930-101; NCT04330534). Here we demonstrate that BCX9930 is a potent and highly selective factor D inhibitor, and completely blocked AP activation on PNH erythrocytes in vitro. METHODS: Factor D enzymatic activity was analyzed in vitro in an esterolytic assay with a synthetic substrate and in a proteolytic assay using its natural substrate factor B bound to C3b. The selectivity was assessed in substrate-specific assays using other human serine proteases. The inhibition of AP-mediated hemolysis was assessed in vitro with rabbit erythrocytes incubated with 10% normal human serum (NHS). The Ham test was used to evaluate the inhibition by BCX9930 of AP-mediated hemolysis of erythrocytes from PNH patients with 20% acidified NHS. Inhibition of AP-mediated C3 fragment deposition was evaluated by flow cytometry using PNH erythrocytes incubated with acidified C5-depleted NHS. Activity of the classical pathway (CP) of complement l was evaluated by measuring 50% Hemolytic Complement (CH50) activity of serum and by assay of CP-mediated hemolysis of antibody-sensitized sheep erythrocytes. Pharmacodynamic effects in rhesus monkeys dosed orally with BCX9930 (100 and 200 mg twice-a-day) were assessed ex vivo with the AP Wieslab assay for the determination of C5b-9 generation after AP activation. RESULTS: BCX9930 potently inhibited esterolytic activity of purified human factor D with a mean 50% inhibitory concentration (IC50) of 14.3 nM. BCX9930 also inhibited its proteolytic activity against factor B bound to C3b with a mean IC50 of 28.1 nM. BCX9930 inhibited AP-mediated hemolysis of rabbit erythrocytes with a mean IC50 of 29.5 nM. Using the Ham test with erythrocytes from three PNH patients, BCX9930 completely suppressed AP-mediated hemolysis with a mean IC50 of 35.4 nM. Furthermore, BCX9930 suppressed C3 fragment deposition on PNH erythrocytes with a mean IC50 value of 39.3nM. BCX9930 did not inhibit enzyme activity of the human serine proteases thrombin, activated protein C, tissue plasminogen activator and trypsin up to 28 μM, and activated factor X and activated factor XII up to 50 μM. The mean IC50 values of BCX9930 inhibition of C1s and plasmin were 936 and 2850 nM, respectively. BCX9930 at a concentration up to 3 μM had no effect on CP activity in in vitro assays. Oral dosing of BCX9930 in monkeys completely suppressed AP activity of serum measured by AP-dependent generation of C5b-9. CONCLUSIONS: BCX9930 inhibitory activity on Factor D in vitro was potent and highly specific. BCX9930 completely blocked hemolysis of PNH cells in vitro and suppressed the accumulation of C3 fragments on PNH erythrocytes, indicating that BCX9930 has the potential to inhibit both intravascular and extravascular hemolysis. After oral dosing of BCX9930 in rhesus monkeys, AP activity was completely suppressed in ex vivo assays. These data demonstrate that BCX9930, an orally administered investigational drug, is a potent and selective factor D inhibitor, and that targeting factor D with BCX9930 has potential for the treatment of patients with PNH and other alternative pathway mediated diseases. Disclosures Chen: BioCryst Pharmaceuticals, Inc.: Current Employment. Cheng:BioCryst Pharmaceuticals, Inc.: Current Employment. Liu:BioCryst Pharmaceuticals, Inc.: Current Employment. Muppa:BioCryst Pharmaceuticals, Inc.: Current Employment. Parker:BioCryst Pharmaceuticals, Inc.: Current Employment. Zhu:BioCryst Pharmaceuticals, Inc.: Current Employment. Raman:BioCryst Pharmaceuticals, Inc.: Current Employment. Kotian:BioCryst Pharmaceuticals, Inc.: Current Employment. Babu:BioCryst Pharmaceuticals, Inc.: Current Employment.

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.