Procoagulant Activity Research Articles

Phosphatidylserine on Blood Cells and Microparticles Contributes to the Hyper-Coagulable State in Diabetic Kidney Disease

Introduction: Patients with diabetes mellitus (DM) are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially when microvascular complications of diabetes mellitus occur, such as diabetic kidney disease (DKD). Additionally, microthrombosis in glomerular capillaries could decrease the kidney blood flow and induce renal insufficiency. However, patients with DKD do not receive routine antithrombotic therapy. Therefore, more study is needed on the mechanisms of coagulation in DKD with the aim of identifying therapeutic targets. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in the thrombosis. However, relatively little is known about the precise role of PS in the prothrombotic state of DKD. Our objectives were to study the effect of increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin cells in DKD patients, and to evaluate its effect on procoagulant activity (PCA).Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate (UAER), defined as normoalbuminuria (<30 mg/24 h), microalbuminuria (30-299 mg/24 h), or macroalbuminuria (>300 mg/24 h), and compared to healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells and human umbilical vein endothelial cells (HUVECs) were treated with 25 mM, 5 mM or 2.5 mM glucose and then PS exposure was measured. PCA of MPs and cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure and fibrin strands were observed using confocal microscopy.Results: Usingflow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and blood cells (BCs) were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with macroalbuminuria was significantly higher than in patients with normoalbuminuria. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with macro-DKD versus serum from patients with micro-DKD or normo-DKD. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated co-localization of fibrin with PS on ECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 80%. Additionally, both hyperglycemia (25 mmol/L) and hypoglycemia (2.5 mmol/L) induced increased PS exposure on cells compared to normoglycemia (5 mmol/L). Linear regression analysis showed that levels of PS+ BCs and MPs were positively correlated with uric acid and proteinuria, but negatively correlated with GFR.Conclusions: Our results suggested that PS+ MPs and blood cells play a prominent role in inducing the hypercoagulable and prothrombotic state in patients with DKD. The occurrence of abnormal glucose during the hypoglycemic therapy is associated with hypercoagulation in DKD. PS+ cells and MPs likely support thrombosis in renal vascular and result in renal dysfunction and disease progression. Our results demonstrate that PS provides binding sites for FXa and prothrombinase complexes and promotes thrombin formation. Thus, directly targeting FXa and prothrombinase complexes might decrease thrombotic and renal disease risk. The results from this study suggest that future research should focus on discovery and deployment of direct PS inhibitors.Disclosure of Interest: Jialan Shi has a patent for the use of lactadherin as a PS probe. DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies on a Branded Recombinant Factor VIIa and Its Generic Version to Demonstrate Bioequivalence

Background: Recombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for the same indication (AryoSeven™, AryoGen, Tehran-Iran). These generic versions of rFVIIa are claimed to be biosimilar to the Novoseven. The purpose of this study is to compare several batches of generic versions of rFVIIa namely, Aryoseven with multiple commercially available batches of branded Novoseven to determine bioavailability.Methods: Four commercially available random lots of Novoseven were obtained through US and European sources and compared to 5 clinical batches of Aryoseven obtained from Aryogen (Tehran, Iran). These drugs were diluted with saline to make a stock solution of 1mg/ml. Further dilutions were made to obtain working concentrations of 100, 10, 1 and 0.1 µg/ml. Molecular profile using surface enhanced laser desorption ionization (SELDI), gel electrophoretic profile (GEP), immunoblotting studies, and procoagulant activity as measured by clot based method were carried out. Thrombin generation studies were carried out after supplementing with NHP at 0-1.0 ug/ml. The pharmacokinetic profile of one representative batch of Novoseven and Aryoseven was studied in primates after a 50 ug/ml bolus IV in terms of factor VIIa levels.Results: The SELDI mass spectrometric profile of all rFVIIa preparations were comparable. The GEP and immunoblotting studies of the two groups of agents showed a comparable profile with distinct peaks at 50 KDa and 29 KDa. In the clot based functional assays performed in the citrated whole blood and retrieved plasma the two batches of FVIIa produced comparable procoagulant effects in the PT and aPTT assays. In the 0.25 - 1.0 µg/ml range in comparison to the control with saline supplementation, the aPTT did not exhibit any significant shortening whereas there was a sharpening of PT values in the whole blood which was identical in both the AryoSeven™ and NovoSeven® preparations. In the retrieved plasma both products produced comparable effect on the PT and aPTT assays. In platelet rich plasma (PRP) and platelet poor plasma (PPP) AryoSeven and NovoSeven at graded concentration of 0.125 to 1 µg/ml did not produce any measurable change in both PT and aPTT assays. The correction studies carried out in the FVII deficient plasma at a concentration range of 1 to 0.01 µg/ml did not reveal any differences between 2 groups of drugs. The relative correction was comparable in all concentrations for the 2 tissue factor reagents namely Innovin and Recomboplastin 2G. The results were indistinguishable for the 2 groups of drugs. The effect of NovoSeven® and AryoSeven™ on the reversal of anticoagulation in orally anticoagulated patients' were similar in relative correction of the plasma INR for the 2 groups of FVIIa preparations in the range of 0.03 to 1 µg/ml. In the thrombin generation studies both the Novoseven and Aryoseven produced similar effects as measured by increased thrombin generation. In the primate pharmacokinetic studies both groups exhibited similar pharmacokinetic profile.Conclusions: These results demonstrate that four batches of Novoseven and 5 individual clinical batches of Aryoseven were found to be biosimilar. The pharmacokinetic profile of the Novoseven and Aryoseven were studied in primates measuring the factor VIIa level for 6 hours and were found to be comparable. Thus, the generic Aryoseven is biosimilar to branded Novoseven and warrants further validation studies. DisclosuresNo relevant conflicts of interest to declare.

Vascular and Hemostatic Alterations Associated with Pulmonary Hypertension in β-Thalassemia Hemoglobin E Patients Receiving Regular Transfusion and Iron Chelation

Background: β-thalassemia hemoglobin E (β-thal/HbE) is currently the commonest β-thalassemia disease worldwide. Pulmonary hypertension (PH) is the most common cardiovascular complication in this genotype, especially in splenectomized patients. Yet the pathogenesis of this major complication remains poorly characterized. Most of the previous reports investigated pathophysiology of PH in β-thal/HbE with inadequate transfusion. In this study we aimed to define the prevalence and comprehensively explore mechanisms of PH in β-thal/HbE patients receiving regular transfusion and iron chelation which were reported to alleviate PH. Our findings will provide deeper insights into pathogenesis and identify targets for management of persistent PH in thalassemia patients after adequate transfusion and chelation.Methods: β-thal/HbE patients who received regular transfusion ≥ 1 unit per month and any forms of iron chelation over 1 year were eligible for the study. Normal healthy subjects were used as controls. Blood sample collection and echocardiographic assessment were performed on the same days. Patients at risk for PH were defined by the tricuspid regurgitant jet velocity (TRV) ≥ 2.5 m/s.Results: There were 68 β-thal/HbE and 38 healthy controls enrolled between May and September 2015. The ages (mean ± SD) were 34.1 ± 12.6, ranging from 19-67 years, and 37.2 ± 8.6 years, ranging from 18-70 years, respectively. Among 68 β-thal/HbE, the average transfusion duration was 8.1 ± 2.2, ranging from 2.2-10.6 years, and the quantity of transfusion was 22.3 ± 9.6 units per year. The pre-transfusion hemoglobin (Hb) level was 8.1 ± 1.3 g/dL. There were 31 (45.6%) splenectomized patients. β-thal/HbE showed markedly increased erythropoiesis (growth differentiation factor, GDF15, and soluble transferrin receptor, sTfR, levels) and iron load (serum ferritin) compared to controls, all p < 0.001. Thrombin antithrombin (TAT) complex and microparticle's procoagulant activity (MPA) were significantly elevated (both p < 0.001), but total tissue factor antigen (TFAg) and microparticle-associated tissue factor activity (MPTFA) were not different from those of controls, suggesting TF-independent hypercoagulability. Serum nitric oxide metabolites (NOx) and prostaglandin E2 (PGE2) levels were significantly lower than those of controls (both p < 0.001), while soluble thrombomodulin (sTM) levels were significantly elevated (p= 0.045). Serum endothelin-1 (ET-1) and soluble p-selectin levels were not different between patients and controls. Echocardiographic profiles demonstrated preserved biventricular systolic functions in β-thal/HbE with a significant increase in TRV (p < 0.001).Among β-thal/HbE, there were 29 (42.6%; 16 non-splenectomized and 13 splenectomized) cases at risk for PH. Duration and amount of transfusion, pre-transfusion Hb, GDF15 and sTfR levels were not statistically different between PH and non-PH subgroups indicating the similar degree of erythropoiesis. Notably, β-thal/HbE with PH had a significant reduction in NOx levels (p < 0.001) but elevations in TAT, ET-1 and sTM levels compared to those without PH (p= 0.046, 0.005 and 0.026, respectively). Additionally, TRV was significantly correlated with NOx, TAT, sTM and ET-1 levels (r = -0.514, p < 0.001; r = 0.281, p= 0.004; r = 0.313, p= 0.001; and r = 0.245, p= 0.012, respectively). Serum ferritin, MPA, MPTFA, TFAg and PGE2 levels were not statistically different between PH and non-PH subgroups. Surprisingly, there were no differences in any biomarkers and echocardiographic profiles between splenectomized and non-splenectomized patients as well as no significant associations between splenectomy statuses and PH (Χ2 = 0.012, p= 0.914).We reviewed another β-thal/HbE cohort with available flow mediated dilatation (FMD) data. 9 β-thal/HbE with PH had significantly lower FMD (3.43%) compared to 18 β-thal/HbE without PH (6.37%) and 27 age-sex matched controls (9.87%; p= 0.025 and p < 0.001, respectively) indicating a greater degree of endothelial dysfunction in β-thal/HbE having PH. In addition, TRV was significantly correlated with FMD (r = -0.474, p < 0.001).Conclusions: PH is still prevalent in β-thal/HbE patients receiving long-term regular transfusion and iron chelation. In this group, PH is not associated with splenectomy statuses but correlated with NO depletion, TF-independent hypercoagulation and endothelial dysfunction. [Display omitted] DisclosuresSutcharitchan:Chulalongkorn University: Consultancy, Employment; Celgene: Research Funding.

Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing “just in time” prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI-/-), FV deleted (F5-/-) and FV transgenic (F5Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI-/- mice from embryonic lethality.Methods/Results: We performed crossbreeding experiments using F5-/- mice carrying liver- or platelet-specific transgenes. F5LiverTg produces 50% plasma FV with no FV in platelets, whereas F5PltTg produces 3% platelet FV with no FV in plasma. F5-/- F5LiverTgand F5-/- F5PltTgmice were bred into TFPI+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5-/- is embryonic lethal in mice. F5-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5+/- TFPI+/- x F5+/- F5LiverTg TFPI+/-, neither F5-/- TFPI-/- nor F5+/- TFPI-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5+/- TFPI-/- mice, however, it produced grossly normal pups lacking TFPI (F5-/- F5LiverTg TFPI-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5+/- TFPI+/- x F5-/- F5LiverTg TFPI-/-. In this cross, F5-/- F5LiverTg TFPI-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10-12). Next, we bred F5+/- TFPI+/- x F5+/- F5PltTgTFPI+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5-/- F5PltTg TFPI-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5-/- F5PltTg TFPI-/- pups (56 of 291 total mice, p<9.0 x10-18). Both male and female platelet and liver specific F5-/- F5Tg TFPI-/- were fertile, and mothers supported pups until weaning.Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5-/- TFPI-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood. DisclosuresMast:Novo Nordisk: Research Funding.

Interfering with Protein Z Dependent Inhibition of Factor Xa Improves Coagulation in Hemophilia

Blood coagulation involves a highly regulated balance between pro-coagulant and anticoagulant activities, allowing for the abatement of bleeding while limiting clot formation, which, if unchecked, could result in thrombosis. Hemostasis is triggered upon vascular injury when extravascular tissue factor (TF) binds plasma coagulation factor VIIa (FVIIa) forming a FVIIa/TF complex that generates limited amounts of activated factors IX (FIXa) and X (FXa). In normal hemostasis, FIXa with FVIIIa dramatically amplifies the production of FXa; FXa with FVa cleaves prothrombin to thrombin, and thrombin cleaves fibrinogen to form a fibrin clot. FXa generated by FVIIa/TF alone is insufficient for normal coagulation as FVIIa/TF activity is limited due to tissue factor pathway inhibitor (TFPI) and TFPI and other plasma protease inhibitors suppress the action of the initially generated FXa (and thrombin). Lacking FVIII or FIX, hemophiliacs fail to generate the additional FXa that is critical for normal hemostasis. Several methods designed to reduce the activity of endogenous inhibitors of coagulation are currently being investigated as a means to ameliorate the bleeding in hemophilia. These include antibody-mediated reduction in the activities of TFPI and protein S, and siRNA-mediated reduction in antithrombin (AT).Protein Z (PZ) is a vitamin K-dependent coagulation factor that serves as the cofactor for FXa inhibition by the serpin, PZ-dependent protease inhibitor (ZPI), at phospholipid surfaces. If inhibition of the action of PZ/ZPI significantly enhanced coagulation in hemophilia, it might be associated with a reduced risk of thrombosis than other approaches. In the latter regard it is of interest that TFPI, PS and AT deficiency are associated with clinical thrombotic disease and mice lacking these proteins die in utero or perinatally of thromboembolic disease. In contrast, PZ and ZPI KO mice appear healthy.Re-bleeding is a hallmark of clinical hemophilia. A tail vein re-bleeding (TVRB) assay, adapted from the saphenous vein re-bleeding assay of Whinna, was developed to assess the effects of PZ and ZPI deficiency on re-bleeding in the hemophilia A mouse model. In this assay, the lateral vein of the tail (diameter 2.25 mm) of an anesthetized mouse is lacerated to a depth of 1 mm with scalpel and the tail placed in warm saline. When bleeding stops, the wound is wiped with a balled Kimwipe to re-induce bleeding and the tail replaced in warm saline. This process is repeated and the number of wound disruptions occurring within 15 minutes is recorded. In normal mice, the period of bleeding following each wound disruption remains relatively constant, whereas in hemophilia mice the bleeding periods tend to progressively prolong with each subsequent disruption leading to a reduced number of total disruptions over 15 minutes.Disruptions in the mouse TVRB assay were: for C57Bl/6 25.8+1.7 (mean+SEM); for FVIII KO 4.2+1.3; for PZ/FVIII double KOs 15.2+3.2; for ZPI/FVIII doubles KOs 14.6+3.5; for WT/KO littermates 4.0+2.6; and for FVIII KO with 100% human FVIII replacement 25.4+3.7. Based on FVIII dose-response experiments, the results in PZ/FVIII and ZPI/FVIII double KOs were equivalent to ~15% FVIII replacement. This outcome of the TVRB assays was confirmed in tests of the survival of sibling WT/FVIII KO (0%, n=5) and ZPI/FVIII double KO (75%, n=12) mice following tail transection 10 mm from the tip.To assess the translation of the PZ/ZPI results in mice to humans, the effect of an anti-PZ monoclonal antibody (Mab) was compared to FVIII replacement on TF (1 pM) induced thrombin generation in human hemophilia A plasma. The Mab enhanced thrombin generation in hemophilia plasma to a similar degree as ~15% FVIII.These studies provide proof of concept, both in vivo in mice and in vitro in human plasma, that blockade of the PZ/ZPI system may be sufficient to alter the phenotype of severe hemophilia to that of mild hemophilia, perhaps with limited thrombotic risk. DisclosuresNo relevant conflicts of interest to declare.

Platelet Tissue Factor Activity Is Specifically Induced By Gpib Activation Pathway, Independent of GPVI, αIIbβ and Protein Disulfide Isomerase Activities

Megakaryocyte-synthesized Tissue Factor (TF) is transferred to platelets and carried “encrypted” in circulation. Platelet glycoprotein receptors, including GPIb and αIIbß3 integrin contain thiol groups representing redox sensitive sites. PDI is externalized and exposed on platelet membranes during the secretion process. Moreover, integrin ß subunits contain sequences with intrinsic PDI activity, as well as vicinal thiol groups which, in ß3 appear to regulate platelet activation. Several reports assign a mechanistic role to thiol isomerases in de-encrypting and activating TF in other cells, but the role of PDIs in platelet TF activation has not yet been explored. The extracellular TF domain proximal to the membrane contains Cys186-Cys209 disulfide, which is a likely target for PDIs. GPIb also has redox sensitive thiol groups. We have reported that VWF-induced GPIbα activation induces platelet TF activation (Atherosclerosis 2017;257:164). Now, we studied the role of PDI, GPVI and αIIbß3 in platelet TF activation. We tested polyclonal and RL-90 mAb α-PDI, as well as various PDI inhibitors (rutin, purified bacitracin, PACMA and 16F16), all of which inhibited aggregation and secretion of washed human platelets by TRAP, but not by VWF-Ristocetin (Fig A). The number of platelets binding Annexin V (flow cytometry) increased from ≈2-3% to >60% with both agonists. Basal platelet TF activity (24nM 2x10-7platelets) increased by 6-fold after stimulation with VWF-Ris, whereas ADP, TRAP and Collagen had no effect on FXa generation (Fig C). None of the PDI inhibitors had significant effect on FXa induced by VWF-Ris activation (Fig D). Membrane fractions obtained from thiol biotinylated platelets and IP with α-TF showed co-IP with PDI and TF thio-biotinylation, more pronounced after stimulation. This effect was not selective for VWF-Ris or TRAP.VWF-Ristocetin stimulated platelets from 4 unrelated patients with homozygous GPVI deficiency (J Thromb Haemost 2013;11:1751) had normal generation of FXa (Fig E). Two patients with Glanzmann disease (Fig F) and 8 samples of washed platelets obtained from blood drawn with tirofiban added to the anticoagulant solution (Fig G) had very low FXa generation in resting platelets, which increased up to 100-fold after VWF-Ris stimulation. No FXa was induced by collagen or TRAP activation.Conclusions. These observations strongly suggest that platelet PDI (and possibly PDI from other sources) is not involved in platelet TF activation and that PDI does not interfere with GPIbα function. Other mechanism(s) must be explored to explain the initiation of clotting by platelet TF. Moreover, our results highlight the specificity of GPIbα activation pathway in inducing physiological TF procoagulant activity, since neither GPVI nor αIIbß3 play a significant role in the clotting function of human platelets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tissue Factor-Related Biomarkers and Prediction of Cancer-Associated VTE: Correlative Analyses of the Phacs Trial

Background: Cancer patients at high or low risk for venous thromboembolism (VTE) can be predicted by a validated score (Khorana et al, Blood 2008) but the mechanisms underlying this association are not fully understood. We evaluated the association of VTE-related biomarkers with clinical outcomes in patients enrolled in a randomized trial of outpatient prophylaxis in high-risk patients and a control population of low-risk patients.Methods: Cancer patients with risk score ≥3 starting systemic therapy were randomized to dalteparin or observation for 12 weeks; a low-risk group of controls (score=0) was concurrently enrolled. Analyses for tissue factor (TF) using microparticle procoagulant activity, TF pathway inhibitor (TFPI) using ELISA, D-dimer and factor VIIA were conducted on specimens obtained at enrollment and 4-weekly on study as well as at enrollment in control patients.Results: The study populations comprised 89 randomized high-risk patients and 101 low-risk patients. TF MP-PCA levels were more likely to be detectable in high-risk patients (51.7% vs 32.7% of low-risk patients, p=0.01) and levels were elevated at time of enrollment in high-risk compared to low-risk patients (mean 0.67 vs 0.19 pg/mL in low-risk, p=0.002; median 0.03 vs 0 pg/mL, p=0.001). TF levels were particularly high in the subgroup of high-risk patients with pancreatic cancer (N=38, 57.9% detectable, mean 0.87 pg/mL, median 0.13 pg/mL). D-dimer levels were also significantly elevated in high-risk patients (median 1.23 vs 0.92 mg/mL, p=0.006). Levels of F1.2 and FVIIa were not significantly different between the high- and low-risk groups. Patients randomized to dalteparin had a significant decline in median D-dimer levels (median 1.22 mg/mL for timepoint 1 versus 0.7 mg/mL for timepoint 4, p =0.018) whereas there was no significant change over time for patients randomized to observation (median 1.30 vs 1.33 mg/mL, p=0.43). F1.2 levels also declined over time for patients randomized to dalteparin (median 77.17 to 32.37, p=0.01) but not for patients randomized to observation (median 59.06 to 61.63, p=0.99). In multivariate time-dependent Cox analysis in high-risk patients, VTE was associated with both doubling of TF (HR 1.42 95% CI 1.17-1.84, p=0.0003) and doubling of TFPI (HR 3.21, 95% CI 1.14-9.50, p=0.02).Conclusions: Cancer patients at high-risk for VTE are more likely to have increased TF levels at baseline; increased levels of both TF and TFPI were associated with future VTE. Further investigations into the role of TF and TFPI in cancer-associated thrombosis and their roles as predictive biomarkers are warranted. DisclosuresKhorana:Pfizer: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Leo: Consultancy, Honoraria, Research Funding. Francis:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

Genetic Fusion to Factor XIII B-Subunit Improves the Pharmacokinetic Properties of Factor IX

Background: Hemophilia B is a X-linked recessive bleeding disorder characterized by coagulation factor IX (FIX) deficiency. FIX concentrates are used in the prevention and treatment of bleeding episodes in patients with hemophilia B. Prophylaxis is currently considered as the optimal care for severe hemophilia. For patients and their families one of the major problems with prophylaxis is the need for frequent venepunctures. The half-life of standard FIX concentrates is approximately 18 h, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life FIX concentrates have been developed using two major strategies: i) (glyco) PEGylation(1), ii) genetic fusion to immunoglobulin Fc fragments(2), or to albumin(3). These technologies may have some potential disadvantages such as reduced specific activity, unknown long-term adverse effects of PEG exposure and immunological reactivity which are currently under investigation.The aim of the present study was to develop a new strategy to prolong half-life of recombinant human FIX molecule (rFIX) using an innovative approach of fusion.Factor XIII (FXIII) is a protransglutaminase that circulates in plasma as a heterotetramer composed of two catalytic A subunits and two B subunits (FXIIIB). FXIIIB subunits act as a carrier protein that stabilizes the structure of the A subunits and protects FXIII from proteolysis. It has been reported that a primary deficiency of FXIIIB resulted in the secondary rapid loss of A subunit from the plasma (4). Thus, FXIII-B is responsible for the long half-life of the FXIII molecule (10 to 12 days).We designed a new rFIX molecule fused to coagulation FXIII subunit B (FXIIIB) and separated by a short linker sequence containing an activated Factor X-cleavable site (rFIX-LXa-FXIIIB). Thus, we expected that the carrier protein will be removed as soon as traces of (activated factor X) FXa will be generated, leaving rFIX free to perform its enzymatic role in the tenase complex.Methods: Recombinant FIX-LXa-FXIIIB molecules were expressed in human hepatic Huh-7 cell line and purified via a two-step protocol prior to in vitro and in vivo biochemical and pharmacokinetic characterizations.Results: rFIX-LXa-FXIIIB was correctly expressed by Huh-7 cells at the expected molecular weight of 150 kDa and presented similar procoagulant activity compared to rFIX-WT (106.5 UI/dL ; p> 0.05). We showed that the LXa linker was cleavable by FXa, releasing free rFIX molecule before its activation. After cleavage, rFIX was fully activable by activated FXI. Both rFIX-WT and rFIX-LXa-FXIIIB showed similar thrombin generation capacity both in vitro and in vivo after injection in FIX-deficient mice (Area under the thrombin generation curve =300nM.min ; p>0.05).The half-life (T1/2) of rFIX-LXa-FXIIIB molecule was determined in wild-type mice and rats. In mice, as commercially available extended half-life FIX products, the improvement of the half-life was very limited and in rats, it was 2.5 fold longer compared to rFIX-WT (T1/2 10h vs 4h ; p<0.05).A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIIIB. It has been recently reported that excess plasma FXIIIB2 is not free, but rather circulates bound to fibrinogen (5). We can hypothesize that the rFIX-LXa-FXIIIB fusion protein can circulate bound to fibrinogen and might therefore enhance/accelerate the transformation of fibrinogen into fibrin. In vivo experiments using mice saphenous vein bleeding model are currently conducted in our laboratory to test this hypothesis.Conclusion: We report here a new fusion protein strategy using FXIIIB. The fusion protein has normal specific activity and normal thrombin generating capacity, associated with improved pharmacokinetics in small animal models. These encouraging results suggest that genetic fusion of rFIX to FXIIIB might be a valuable concept to improve the half-life of FIX molecules. The binding capacity of this novel fusion protein to fibrinogen might be an additional advantage that may improve haemostatic capacity of the molecule.1. Negrier C et al. Blood. 2011;118:2695-701.2. Peters RT et al. Blood. 2010;115:2057-64.3. Metzner HJ et al. Thromb Haemost. 2009;102:634-44.4. Miloszewski K et al. Br J Haematol. 1970;19:685-90.5. Byrnes JR et al. Blood. 2016 Oct 13;128:1969-1978 DisclosuresLe Quellec:LFB: Honoraria; Novo Nordisk: Honoraria; Shire: Honoraria. Negrier:Baxter: Research Funding; Alnylam: Research Funding; Bayer: Honoraria, Research Funding, Speakers Bureau; Biogen/SOBI: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Inspiration: Research Funding; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Octapharma: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau; LFB: Honoraria, Speakers Bureau. Dargaud:BMS: Honoraria, Research Funding; Baxalta/Shire: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; LFB: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Stago: Honoraria; Octapharma: Honoraria, Research Funding; Alnylam: Research Funding.

Procoagulant Mechanisms in Patients with Myelodysplastic Syndrome

Background: MDS comprise a heterogeneous sort of disorders caused by cytogenetic changes, gene mutations, or both, responsible for altered multilineage morphological patterns that lead to a varying grade of peripheral blood cytopaenias and an increased risk of transformation to acute myeloid leukemia. Thrombocytopaenia is observed in aproximately 35% of MDS patients. Despite it is accepted that haemorrhagic events are more likely at platelet counts below 10,000/μl, in the case of MDS patients, whose platelets had a diminished ability to be activated, bleeding events are lower than expected. So, hemostasis in MDS patients is a complex issue where mechanisms additional to platelet count/function seem to be involved.Aim: This work aimed to identify procoagulant mechanisms in non-bleeders MDS patients that might protect them from bleeding.Methods: This is a prospective, observational and transversal study. Eighty four non-bleeders MDS patients (44% female, mean age of 77±25 years), were included. Sixty healthy subjects (45% female, mean age of 57±22 years) were recruited.Human peripheral blood samples were collected in 3.8% sodium citrate. Blood cell counts were performed with a Coulter Ac.T Diff cell counter (Beckman Coulter, Madrid, Spain). Platelet rich plasma (PRP) and platelet free plasma (PFP) were obtained by centrifugation.Surface exposure of phosphatidylserine (PS) and active caspase-3, -8 or -9 were assessed by flow cytometry measuring the binding of FITC-labeled Annexin V (BD Pharmingen) and with a specific kit for measuring these caspases (Millipore, Madrid, Spain).Prothrombinase complex binding to platelets was determined in quiescent washed platelets (1x108/mL) incubated with factor (F) Xa (5 nmol/L) and FVa (5nmol/L) obtained from Haemtech Inc (Cellsystems, Germany). Following fixation to cross-link platelet-bound FVa and FXa, specific or control monoclonal antibodies (0.1 μmol/L each, Haemtech Inc, Germany) were added. All samples were analized by flow cytometry.PS-associated and tissue factor (TF)-associated procoagulant activity of microparticles (MPs) was determined with the ZYMUPHEN MP-Activity kits (HYPHEN BioMed, Neuvillesur Oise, France).The identification of the MP's cellular origin was determined by flow cytometry, labeling MPs with Annexin-V-FITC and the following specific mAb conjugated with phycoerythrin: anti-CD41 mAb for platelets (Biocytex, Marseille, France); and anti-CD14 mAb for monocytes, anti-CD144 mAb for endothelial cells, anti-CD235 mAb for red cells and anti-CD45 mAb for leukocytes, all from BD Biosciences.The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelets from patients with MDS presented higher levels of activated caspases-3,7, -8 and -9 and this situation was accompanied by a higher PS platelet surface exposure (TABLE 1)We evaluated if increased PS exposure was functional for anchoring prothrombinase complex. Binding of FVa and of FXa on platelet membrane surface was higher in quiescent platelets from MDS patients [FVa (Mean fluorescence), control: 49.7±8.5, MDS patients: 73.9±23.2, p<0.01; FXa (Mean fluorescence), control: 46.4±17.5 , MDS patients: 77.6±12.0, p<0.05)].A significantly higher procoagulant capacity of MPs associated either to PS (control: 8.0±2.5 nanomol/L; MDS: 17.8±3.5 nanomol/L, p<0.01) or TF (control: 0.4±2.5 nanomol/L; MDS: 7.5±2.5 nanomol/L, p<0.001) were observed in the MDS group.To determine the cellular source of MPs, flow cytometry analyses were performed and a significant increase was observed in the amount of monocyte- (events, control: 94±30, MDS patients: 220±29, p<0.05)and red cell-derived MPs (events, control: 142±55, MDS patients: 252±23, p<0.01) in the samples from the patients with MDS. In patients with MDS, TF-MP-associated thrombogenic ability correlated with monocyte percentage (Spearman r=0.5019, p<0.01).Conclusion: MDS patients, even those with normal platelet count, have a reduced platelet function with enhanced apoptosis but do not bleed in many cases. This fact might be due to the increase in tissue factor-containing microparticles and in the enhanced binding of prothrombinase complex to apoptotic platelets which, in turn, augmented procoagulant capacity of these patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresJimenez-Yuste:Roche: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding. Álvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy.

FVIII Proteins with a Modified Immunodominant T-Cell Epitope Exhibit Reduced Immunogenicity and Normal Procoagulant Activity

Factor VIII (FVIII) neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 (legacy FVIII numbering, sequence: SYFTNMFATWSPSKARLHLQ) has been shown to contain an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells isolated from HA subjects. The aim of this study was to identify amino acid substitutions to de-immunize this epitope while retaining pro-coagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The minimal DRB1*01:01-binding motif was mapped to FVIII2194-2205 by quantitative peptide-MHC Class II (MHCII) binding assays. Further binding assays with sequence-modified FVIII peptides established that the residues important for DRB1*01:01 affinity were F2196, M2199, A2201, and S2204, corresponding to MHCII anchor positions 1, 4, 6 and 9. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation and cytokine secretion of five clones specific for FVIII2194-2213. The substitution F2196A was then engineered into a recombinant (r)FVIII-C2 protein. T-cell clones that were strongly stimulated by wild-type rFVIII-C2 did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic synthetic peptide includes a naturally processed T-cell epitope. The measured and predicted effects of additional amino acid substitutions within this binding motif were further evaluated by peptide-MHCII binding assays, CD4+ T-cell proliferation assays, and MHCII-binding prediction algorithms. Amino acid homologs within this binding motif were evaluated with sequence homologies to FVIII and FV proteins from different species. On the basis of all these results, six rB-domain-deleted (BDD)-FVIII proteins with amino acid substitutions F2196A, F2196L, F2196K, M2199A, M2199W or M2199R, respectively, were designed and produced; all but the F2196A variant had specific activities comparable to that of rWT-BDD-FVIII. The F2196A variant also had the lowest expression level among the rBDD-FVIIII proteins produced. Peptide-MHCII binding assays confirmed that FVIII2194-2205 peptides with substitutions F2196A, F2196K, M2199A and M2199R did not bind to DRB1*01:01 or to an additional 9 HLA-DR proteins tested, with the exception of the M2199A peptide binding to DRB1*09:01. The predicted effects of these FVIII sequence modifications on binding to additional MHCII alleles included in reference sets representing ~99% of the human population HLA diversity were evaluated using ProPred and Immune Epitope Database algorithms. These programs correctly predicted that the substitutions would abrogate binding to DRB1*01:01 and also indicated that affinities for additional MHCII would be reduced by each substitution. The substitutions were predicted to increase peptide-MHCII affinities for a smaller number of MHCII alleles, indicating a possible risk of creating neo-epitopes restricted to these specific HLA alleles. Importantly, proliferation of patient-derived T-cell clones in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A proteins was dramatically reduced compared with responses to rWT-BDD-FVIII. We have previously demonstrated that a severe HA inhibitor subject had an oligoclonal T-cell response to only one T-cell epitope within the FVIII A2, C1 and C2 domains and that one epitope was FVIII2194-2213 (Ettinger et al., Blood 2106; 128(16):2043). This observation, together with the results presented here, further suggests that modification of only a small number of immunodominant epitopes may be sufficient to significantly de-immunize FVIII and decrease FVIII inhibitors in HA patients. The present results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients. DisclosuresEttinger:Bloodworks NW: Patents & Royalties: Inventor on FVIII patents. James:Bloodworks NW: Patents & Royalties: inventor on FVIII patent(s). Pratt:Bloodworks NW: Patents & Royalties: inventor on FVIII patents.

Improved Inhibition of Protease-Activated Receptor 4 (PAR4) with Function-Blocking Antibodies Provides Equivalent Antithrombotic Activity in the Face of the Hyper-Reactive Thr120 PAR4 Variant

Background: Thrombin activates platelets via two protease-activated receptors (PARs), PAR1 and PAR4, and both are targets of antithrombotic drugs - a PAR1 inhibitor is approved for clinical use and PAR4 inhibitors are in trial. However, recent studies report a PAR4 variant (rs773902; encoding Thr120 instead of Ala120) is expressed in up to 80% of individuals, depending on the population, and renders the receptor more sensitive to agonists and less sensitive to antagonists. These studies suggest the effectiveness of current strategies to inhibit PAR4 may vary substantially across the population. Here, we report the generation of a novel function-blocking PAR4 antibody that provides equivalent antithrombotic activity against the Ala120 or Thr120 PAR4 variant.Methods: We generated a panel of anti-PAR4 antibodies and examined their inhibitory activity against the Ala120 and Thr120 PAR4 variants in cultured cells, isolated platelets, and in an ex vivo whole blood thrombosis assay.Results: Our lead function-blocking antibody bound human PAR4 with high selectivity (~16-fold over PARs1, 2 or 3) and affinity (KD ~ 5nM). This antibody provided near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant when transfected in HEK293T cells. Platelets isolated from individuals expressing the Thr120 PAR4 variant exhibited increased thrombin-induced aggregation and phosphatidylserine exposure versus those expressing the Ala120 PAR4 variant. Yet the anti-PAR4 antibody inhibited these responses with an equivalent IC50 of ~ 5 µg/ml. Furthermore, this function-blocking anti-PAR4 antibody significantly impaired platelet procoagulant activity in an ex vivo whole blood thrombosis assay. Here, equivalent inhibition of thrombin generation (FRET-based thrombin substrate) and fibrin formation (anti-fibrin antibody) was observed in thrombi generated in blood from individuals expressing the Ala120 or Thr120 PAR4 variant.Conclusion: We generated a highly selective and effective function-blocking anti-PAR4 antibody that impairs thrombin-induced PAR4 cleavage, platelet activation, and pro-thrombotic activity equivalently in the presence of the Ala120 or Thr120 PAR4 variant. These findings reveal a novel approach to PAR4 inhibition that overcomes the impaired sensitivity of existing approaches against the commonly expressed Thr120 PAR4 variant, and provide rationale for such an approach for improved antithrombotic therapy. DisclosuresNo relevant conflicts of interest to declare.

Alteration of Endothelial Cell Hemostatic Equilibrium By Cancer Cell-Derived Microparticles, a New Insight in Pathogenesis of Cancer Associated Thrombosis

Background: Cancer is associated with hypercoagulable state and frequently complicated by venous thromboembolism (VTE). Microparticles (MPs) are implicated in the pathogenesis of cancer associated thrombosis. However, not much is known about their interaction with the endothelium.Aim: To study the effect of cancer cell derived MPs on the hemostatic balance of endothelial cells.Methods: Microparticles released in conditioned medium from pancreas adenocarcinoma cells (BXPC3) and human breast cancer cells (MCF7) were isolated by differential centrifugation from cell cultures at 90% confluence. The pellets were washed twice in phosphate-buffered saline (BSA) and re-suspended in 1 ml Endothelial cell Basal Medium (EBM-2). Human umbilical vein endothelial cells (HUVEC) were cultured in 96-well plates in presence or absence of MPs derived from BXPC3 or MCF7 cells for 72h (C0). Then they were washed twice in BSA. and re-suspended in serum RPMI-1640 medium and re-cultivated over three cycles (C1-C3) following the same procedure of washing and culture.Thrombin generation (TG) of normal platelet poor plasma (PPP) added in wells carrying HUVEC were assessed by the Calibrated Automated Thrombogram (Rousseau et al Thromb Res. 2015;136:1273-9). Furthermore, TF activity (TFa) was assessed with a clotting based assay (from Diagnostica Stago, France). Cells and MPs were analyzed by flow cytometry for TF and annexin V expression using specific antibodies.Results: Some traces of TFa were found in HUVEC cells (0.20±0.03pM) and HUVEC MPs (1.2±1.0pM) being comparable to normal PPP (0.19±0.02pM). Abundant amounts of TFa were found in BXPC3 cells (2.6±0.05pM) and BXPC3 MPs (920±50pM). MCF7 cells expressed low amounts of TFa (1.21±0.05pM) whereas TFa levels in MCF7 MPs were significantly higher (12.1±2.2pM). Flow cytometry analysis for TF expression showed similar results for the three types of cells and the corresponding MPs. No procoagulant phospholipids were found on the surface of the three cell lines. In contrast, procoagulant phospholipids were expressed by BXPC3 and MCF7 MPs but not by HUVEC derived MPs. HUVEC exposed to cancer cell derived MPs enhanced thrombin generation. This procoagulant property of HUVEC was acquired after exposure to MPs and remained significant after three cycles of cells culture. MPs derived from BXPC3 had more pronounced effect on HUVEC as compared to MPs derived from MCF7 (Table 1).Conclusion:Cancer cell-derived microparticles possess significantly higher procoagulant activity as compared to the cells of origin. They are characterized by high expression of TF and procoagulant phospholipids. Cancer cell-derived MPs induced shift of HUVEC hemostatic balance. Quiescent HUVEC were transformed to potent procoagulant cells and enhanced thrombin generation. The procoagulant properties acquired by HUVEC are transferred to the following cells generations. To the best of our knowledge, the present study shows for the first time that the inherent procoagulant properties of MPs could alter vascular functions. The impact of MPs on the procoagulant phenotype of the HUVEC varies according to the type of cancer cell line. Overall, the findings of the present study provide novel information regarding the specific contribution of tumor-derived MPs to the pathogenesis of cancer-associated thrombosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Optimization of Clot Formation Under Blood Flow in a Tissue Engineered Blood Vessel

BackgroundHemostasis is a complex event relying on the intersection of coagulation and platelet plug formation at the damaged blood vessel wall. Blood flow is an important force during hemostasis that delivers and removes reactants and cells to the growing clot. Standard in vitro plasma-based clotting assays omit the influence of blood flow, blood cells, and the damaged vessel wall. To address this gap, we are developing a model of laser-induced clot formation under flow, in tissue engineered blood vessels, utilizing human proteins and cells. Our goal is to develop a system allowing the study of coagulation and platelet accumulation under physiological flow and cellular environments.MethodsBlood vessels (150 um in diameter) were formed in microfluidic devices by seeding endothelial cells in a tubular void within a collagen I matrix and culturing under flow conditions. A variety of human microvascular endothelial cells types were tested. Normal human donors provided blood with informed consent. To measure fibrin and platelet accumulation, blood was spiked with fluorescently-labelled fibrinogen and calcein-AM. Laser ablations were performed with a 532 nm pulse laser and time lapse images of clot formation were captured using widefield fluorescence microscopy.ResultsWhile most endothelial cell types tested could form blood vessels, only brain endothelial cells, with supporting brain pericytes seeded in the collagen I matrix, were able to reproducibly form blood vessels capable of supporting blood flow without leakage. Brain endothelial cell blood vessels could support blood flow for greater than 45 minutes. Upon laser ablation, and after occasional red blood cell leakage, fibrin(ogen) and platelet accumulation occurred at the site of injury. Clots began forming shortly after ablation (2.1 ± 1.2 minutes until platelet accumulation and 0.5 ± 0.4 minutes until fibrin(ogen) accumulation) and maximum platelet accumulation was reached 12.3 ± 3.2 minutes post-ablation. Overall clot architecture mirrors that reported with in vivo laser injury models where fibrin(ogen) accumulates at or outside the vessel wall and platelets form a primary plug with limited intravascular accumulation.ConclusionsTissue engineered blood vessels composed of brain endothelial cells with supporting pericytes in microfluidic devices can support blood flow and laser-induced clot formation. Clots that form have a similar architecture to clots formed in vivo . This model, which combines blood plasma and cells with blood flow and the damaged vessel wall, has the potential to facilitate investigation of the procoagulant activity of hemostatic drugs in a physiologic, all human system. DisclosuresKistanova:Bioverativ: Employment, Equity Ownership. Aleman:Bioverativ: Employment, Equity Ownership. Salas:Bioverativ: Employment, Equity Ownership.

Bioresponsive starPEG-heparin hydrogel coatings on vascular stents for enhanced hemocompatibility

Hydrogel coatings can improve the biocompatibility of medical devices. However, stable surface bonding and homogeneity of hydrogel coatings are often challenging. This study exploits the benefits of biohybrid hydrogels of crosslinked four-armed poly(ethylene glycol) and heparin to enhance the hemocompatibility of cobalt‑chromium (CoCr) vascular stents. A bonding layer of dual silane and poly(ethylene-alt-maleic anhydride) (PEMA) treatment was applied to the stent to provide covalent immobilization and hydrophilicity for the homogeneous spreading of the hydrogel. A spray coating technology was used to distribute the aqueous solution of the reactive hydrogel precursors onto the sub-millimeter struts of the stents, where the solution polymerized to a homogeneous hydrogel film. The coating was mechanically stable on the stent after ethanol dehydration, and the stents could be stored in a dry state. The homogeneity and stability of the coating during stent expansion were verified. Quasistatic and dynamic whole blood incubation experiments showed substantial suppression of the pro-coagulant and inflammatory activity of the bare metal by the coating. Translation of the technology to industrial coating devices and future surface modification of stents with anti-inflammatory hydrogels are discussed.

An ultrasmall infinite coordination polymer nanomedicine-composited biomimetic hydrogel for programmed dressing-chemo-low level laser combination therapy of burn wounds

To programmatically promote burn wound healing in a way that was tailored to the complex healing phases of hemostasis, anti-inflammation, proliferation, and remolding, we proposed a combined therapeutic strategy of dressing management-chemotherapy-low level laser therapy (LLLT), and designed a highly integrated and simply-structured nanocomposite hydrogel with properties of transparency, self-monitored pH-responsive drug release, and burn wound-adaptability (shape-remodeling, self-healing, and tissue-adhesion). This nanocomposite hydrogel was composed of oxidized hyaluronic acid (OHA), ε-poly-L-lysine grafted human-like collagen (HLC-EPL) and a versatile curcumin-Fe(III) infinite coordination polymer nanomedicines (Cur-Fe(III) ICPs), which were dual dynamically cross-linked via imine bonds and nanoparticle-polymer interactions. The minimized light scattering and absorption of carrier-free and ultrasmall (9.14 ± 1.25 nm) Cur-Fe(III) ICPs and the low refractive index contrast with water of HLC-EPL and OHA endowed the nanocomposite hydrogel with a light transmittance of ≥ 90.1% at 904 nm, which made it compatible with LLLT. The dynamic crosslinking site-rich surface of the Cur-Fe(III) ICPs improved the shape-remodeling and self-healing efficiency of dressing, which resulted in an 84.6% cross-linking reconstruction efficiency within 10 min, thus making it more adaptive to burn wounds. The programmed therapy was implemented by the autoprogressed intrinsic procoagulant activity of HLC-EPL and physical sealing effect in the hemostasis phase, pH-responsive chemotherapy of Cur-Fe(III) ICPs in the inflammation phase, and spatiotemporally controllable LLLT in the proliferation and remodeling phases. As a result, the closure period of full-thickness burn wounds was greatly shortened from 21 to 9 days, and the reconstruction of skin structure was accelerated.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin.

Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

MicroRNA-340-5p inhibits endothelial apoptosis, inflammatory response, and pro-coagulation by targeting KDM4C in anti-neutrophil cytoplasmic antibody (ANCA)-mediated glomerulonephritis through activation of B cells

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a class of systemic autoimmune diseases, results in damage of various critical organs including kidneys, lungs, eyes, and nervous system. MicroRNA-340-5p was confirmed to be downregulated in autoimmune pathogenesis. However, the role of miR-340-5p remains unknown in ANCA-induced glomerulonephritis (GN). The current study aimed to explore the role of miR-340-5p in ANCA-induced GN. The animal models of ANCA-induced GN was established by experimental autoimmune vasculitis (EAV) operation. The primary glomerular endothelial cells (PGEnCs) were treated with anti-myeloperoxidase (anti-MPO) to mimic cell injury in vitro. The renal function was analysed by measuring serum creatinine, blood urea nitrogen, urine blood, urine protein and urine leukocytes. The levels of RNA and proteins were examined by RT-qPCR and western blot analysis, respectively. The binding capacity between miR-340-5p and KDM4C was detected by luciferase reporter assay. Cell apoptosis was analysed by flow cytometry in vitro. Cell viability was determined by CCK-8 assay. The cleaved caspase-3 activity was analysed by immunofluorescent assay. Cell inflammation was measured by western blot. Cell procoagulant activity was assessed by FXa generation assay. The histological changes of renal tissues were assessed by Haematoxylin and eosin (H&E) staining assay. The correlation between miR-340-5p and KDM4C level (or content of TNF-α and IL-6) was analysed by Pearson correlation analysis. The injection of anti-MPO IgG induced a significant elevation of Serum creatinine and blood urea nitrogen in serum, as well as urine blood, urine protein and urine leukocytes. Importantly, KDM4C was downregulated in model group. In mechanism, we identified that miR-340-5p bound with KDM4C 3'untranslated region (UTR), negatively regulated KDM4C in endothelial cells and negatively correlated with KDM4C in serum of GN rats. In function, we found that miR-340-5p promoted B cell activation and proliferation by downregulating KDM4C. The in vitro assays showed that the decrease of cell viability induced by anti-MPO was reversed by miR-340-5p overexpression, and further reduced by KDM4C overexpression. Inversely, the suppressive effects of miR-340-5p mimics on cell apoptosis, cleaved caspase-3 activity, inflammatory response and pro-coagulation were countervailed by KDM4C overexpression in anti-MPO-treated cells. The in vivo assays validated that miR-340-5p overexpression mitigated the impairment of renal function, and histological changes induced by anti-MPO IgG injection in model group. Finally, we found the negative correlation between miR-340-5p and TNF-α (or IL-6) content in serum of GN rats. In conclusion, we found that miR-340-5p inhibited endothelial apoptosis and inflammatory response by targeting KDM4C in ANCA-mediated GN through activation of B cells, implying a potential novel insight for treatment of ANCA-mediated GN.

7-Ketocholesterol: Effects on viral infections and hypothetical contribution in COVID-19

7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

Neutrophil extracellular traps induce thrombogenicity in severe carotid stenosis.

BackgroundSevere carotid stenosis is a common cause of stroke. In addition, previous clinical studies revealed that patients symptomatic of carotid stenosis suffer from increased episodes of stroke compared with their asymptomatic counterparts. However, the mechanism underlying these differences in the recurrence of stroke remains unclear.ObjectiveThe present study aimed to evaluate the levels of neutrophil extracellular traps (NETs) in the plasma of patients with severe carotid stenosis and investigate whether NETs induced procoagulant activity (PCA) in severe carotid stenosis. The study also sought to investigate the interactions between platelets or endothelial cells (ECs) and NETs.MethodsThe levels of NETs in plasma were quantified using enzyme‐linked immunosorbent assay (ELISA). In addition, NETting neutrophils and neutrophil‐platelet aggregates were detected through flow cytometry. On the other hand, the morphology of NETs formation and endothelial cells were analyzed through confocal microscopy. Finally, the procoagulant activity (PCA) of NETs and endothelial cells were assessed through ELISA and fibrin formation.ResultsPatients with symptomatic carotid stenosis patients had significantly higher levels of NETs markers compared with their asymptomatic counterparts and healthy subjects. In addition, increased levels of neutrophil‐platelet aggregates induced the generation of NETs in patients with symptomatic carotid stenosis. Moreover, NETs contributed to PCA through tissue factor (TF), in patients with carotid stenosis. Furthermore, NETs disrupted the endothelial barrier and converted endothelial cells (ECs) into PCA to enhance the PCA in patients with carotid stenosis.ConclusionsThe current study revealed differences in the levels of NETs in the plasma of symptomatic and asymptomatic patients suffering from carotid stenosis. The study also uncovered the interaction between NETs and thrombogenicity in carotid stenosis. Therefore, inhibiting NETs may be a potential biomarker and therapeutic target for recurring stroke in severe carotid stenosis.

SARS-CoV-2 infection induces the activation of tissue factor–mediated coagulation via activation of acid sphingomyelinase

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte–derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2–SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2–SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2–SP-PV–induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2–SP-PV–induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration–approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.