Blood Coagulation Process Research Articles

Inhibitors of the blood coagulation process in patients with essential thrombocythemia.

: The aim of the study was to assess the activity of protein C, protein S and tissue factor pathway inhibitor in relation to the risk factors for thrombotic complications in patients with essential thrombocythemia.The study group consisted of 45 newly diagnosed patients with essential thrombocythemia. Protein S activity was determined by chromogenic method. Activities of protein C and tissue factor pathway inhibitor (TFPI) were determined using ELISAs.Significantly lower protein C and protein S activity but higher TFPI activity were found in patients with ET in comparison with the control group. TFPI activity was higher in women as compared to men, and in patients over 60 years of age compared with patients below 60 years of age. TFPI activity was higher in patients with leukocytes count at least 11 g/l than in patients with leukocytes count below 11 g/l and the difference almost reached statistical significance. Significantly lower protein C activity was found in patients with the JAK2V617F mutation, in comparison with essential thrombocythemia patients JAK2V617F (-).The reduced protein C and protein S activity may be one of the pathogenic factors of increased prothrombotic state in essential thrombocythemia patients. The decreased protein C activity in patients with the JAK2 V617F mutation seems to confirm the significant role of this mutation in the pathogenesis of thrombotic complications in essential thrombocythemia patients. Significantly increased TFPI activity in essential thrombocythemia patients above 60 years of age and with leukocyte count above 11 g/l expresses the activation of the compensatory mechanism for increased prothrombotic activity.

Existence of pulses for a reaction-diffusion system of blood coagulation

The paper is devoted to the investigation of a reaction-diffusion system of equations describing the process of blood coagulation. Existence of pulse solutions, that is, positive stationary solutions with zero limit at infinity is studied. It is shown that such solutions exist if and only if the speed of the travelling wave described by the same system is positive. The proof is based on the Leray-Schauder method using topological degree for elliptic problems in unbounded domains and a priori estimates of solutions in some appropriate weighted spaces.

Gellable silk fibroin-polyethylene sponge for hemostasis

Traditional haemostatic materials generally have slow hemostasis rate and poor biocompatibility. This paper reports on the haemostatic properties and mechanism of silk fibroin (SF). SF-PEG sponge that could be solubilised and changed to gel form by blood was fabricated through mixing SF and polyethylene glycol (PEG, 1500 Da) followed by lyophilisation of the mixed solution. SF-PEG sponge, together with control samples of SF sponge (no PEG) and a commercially available haemostatic material, gelatine sponge, were subjected to the hemostasis tests using a liver trauma model of rabbit. The results showed that SF was superior to gelatine sponge in hemostasis time (136.17 ± 62.27 s and 249.83 ± 29.18 s) and blood loss (2.16 ± 1.27 g vs. 4.97 ± 1.44 g). Furthermore, in vitro experiments indicated SF-PEG sol-gel transition promoted platelet adhesion and aggregation, as well as platelet-fibrinogen interaction. Therefore, except for the physical blocking of bleeding port due to PEG-induced SF fast gelation, SF might also have an impact on blood coagulation process, a phenomenon that has not been reported before. In conclusion, SF is a new type of haemostatic material that might be able to meet the requirements of speed, efficiency and biosafety in a variety of clinical applications.

Absorbable nanocomposites composed of mesoporous bioglass nanoparticles and polyelectrolyte complexes for surgical hemorrhage control.

Absorbable polyelectrolyte complexes-based hemostats are promising for controlling hemorrhage in iatrogenic injuries during surgery, whereas their hemostatic efficacy and other performances require further improvement for clinical application. Herein, spherical mesoporous bioglass nanoparticles (mBGN) were fabricated, and mBGN-polyelectrolyte complexes (composed of carboxymethyl starch and chitosan oligosaccharide) nanocomposites (BGN/PEC) with different mBGN contents were prepared via in situ coprecipitation followed by lyophilization. The effect of various mBGN content (10 and 20wt%) on morphology, zeta potential, water absorption, degradation behavior and ion release were systematically evaluated. The in vitro degradability was dramatically promoted and a more neutral environment was achieved with the incorporation of mBGN, which is preferable for surgical applications. The in vitro coagulation test with whole blood demonstrated that the incorporation of mBGN facilitated blood clotting process. The plasma coagulation evaluation indicated that BGN/PEC had increased capability to accelerate coagulation cascade via the intrinsic pathway than that of the PEC, while have inapparent influence on the extrinsic and common pathway. The in vivo hemostatic evaluation in a rabbit hepatic hemorrhage model revealed that BGN/PEC with 10wt% mBGN (10BGN/PEC) treatment group had the lowest blood loss, although its hemostatic time is close to that of 20BGN/PEC treatment group. The cytocompatibility evaluation with MC3T3-L1 fibroblasts indicated that 10BGN/PEC induced a ~25% increase of cell viability compared to the PEC at days 4 and 7, indicating improved biocompatibility. These findings support the promising application of absorbable BGN/PEC with optimized mBGN content as internal hemostats and present a platform for further development of PEC-based hemostats.

GLYPROLINE PEPTIDE PHARMACODYNAMICS IN RATS IN THE SETTING OF METABOLIC SYNDROME BY THE PARAMETERS OF LIPID METABOLISM AND TROMBOELASTOGRAMMA

In this study, experimental data on the biological action of glyproline peptides — Pro-Gly-Pro, Pro-ArgPro, Arg-Pro-Gly-Pro and Pro-Gly-Pro-Leu — on the processes of blood coagulation and lipid metabolism were obtained using animal models of metabolic syndrome. Under the conditions of increased blood clotting and hypercholesterolemia, the regulatory peptides under study were administered intranasally for 7 days once every 24 hours. 20 hours after the last peptide administration, thromboelastogram parameters confi rmed the restoration of the functional state of the coagulating and anticoagulant systems. In addition, a simultaneous positive shift in the parameters of lipid metabolism was observed. These changes lasted for 7 days following the withdrawal of the drugs. It is found that the biological action of the peptides under study consists in their simultaneous regulatory impact on the processes of hemostasis and lipid metabolism.

Procoagulant Microparticles Derived from Myeloma Plasma Cells Have a Determinant Role in the Hypercoagulable State Associated with Multiple Myeloma. a Modelization in Vitro Study

Background: Multiple myeloma (MM) is a plasma cell malignancy characterized by bone marrow infiltration leading to multiple lytic bone lesions, renal failure, anemia and increased rate of thrombotic events. The cross-talk between cancer cells, plasma clotting mechanism, platelets and endothelial cells enhances hypercoagulability. The mechanisms by which myeloma plasma cells (MPCs) interfere with their microenvironment during the blood coagulation process have been poorly investigated. Several studies have established a link between levels of cancer cell derived procoagulant microparticles, cancer aggressiveness, response to anticancer treatments well as with the risk of cancer associated thrombosis., The mechanisms by which myeloma plasma cells interfere to blood coagulation process in their microenvironment have been poorly investigated. Aim: Set up an experimental model that allows the identification of the procoagulant fingerprint of MPCs and myeloma cell-derived microparticles (MC-dMPs) as well as their impact on thrombin generation of normal plasma. In addition, we evaluated the relative impact of TF-pathway and the intrinsic clotting pathway on thrombin generation triggered by myeloma plasma cells and MC-dMPs. Finally, we performed a simulation of thrombin generation process in the presence of myeloma plasma cells and MC-dMPs as well as in the presence of varying concentrations of TF and procoagulant phopsholipids, mimicking some of the conditions that potentially exist in MPCs. Methods. TF and annexin V expression by MPCs (MPC-dMPs) were analyzed by flow cytometry, TF activity (TFa) and TF gene expression was also determined. Thrombin Generation (TG) in the presence of MPCs or MPC-dMPs was assessed with the Calibrated Automated Thrombogram assay (CAT®) in normal human PPP. TG was also assessed in plasma spiked with MPCs and MPC-dMPs or variable concentrations of TF and procoagulant phospholipids. Results: MPC-dMPs expressed about 8-fold higher levels of TF as compared to MPCs. TFa expressed by MPC- dMPs was significantly higher as compared to that expressed by MPC cells. MPCs and PC- dMPs enhanced thrombin generation of human plasma. Thrombin generation was significantly higher with MPC-dMPs compared to MPCs. MPCs and MPC-dMPs, when concomitantly present in normal PPP, significantly amplified thrombin generation as compared to that observed in the presence of MPCs alone. Presence of TF and procoagulant phospholipids in the microenvironment resulted in significant amplification of thrombin generation induced by MPCs. Conclusion: This study support the concept that hypercoagulability induced by myeloma plasma cells is the result of the combination of the procoagulant properties of myeloma cells with the procoagulant elements of the plasma microenvironment which consist of procoagulant phospholipids and low TF concentration. Following this concept we assume that MPC-dMPs released by myeloma plasma cells into the microenvironment enhance the weak procoagulant activity of myeloma plasma cells. Whereas this combined effect of MPCs and MPC-dMPs on thrombin generation has an impact on the formation of local fibrin network their critical role into the hypercoagulablity, procoagulant MC-dMP could be a potential target for the evaluation of the aggressivity of MPCs and of their response to anti-myeloma treatment. Disclosures Dimopoulos: Sanofi Oncology: Research Funding. Mohty:Jazz Pharmaceuticals: Honoraria, Research Funding. Elalamy:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; LEO Pharma: Honoraria, Research Funding; Aspen: Honoraria, Research Funding; Bayer Healthcare: Honoraria, Research Funding.

Effect of Sulfated NON-Anticoagulant Low Molecular Weight Heparin in Cancer and NON-Cancer Associated Thrombosis

Introduction: The process of thrombosis depends on a complex interaction between different factors including vascular endothelial activation, platelet activation and adhesion. Blood coagulation process initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. Cancer-associated thrombosis (CAT) accounts for about 20% of all cases of Venous Thromboembolism (VTE). Tissue factor (TF) is documented to be highly expressed on the cancer cells and pathological angiogenic endothelial cells. Subsequent theories proposed that the cancer cells activate the coagulation cascade, as the oncogenes cause tumor transformation, which in turn causes the cancer tissues to be able to express different pro-coagulant proteins. VTE risk factors can be divided into cancer- and therapy-associated factors. Low molecular weight heparin (LMWH) is used for prophylaxis and treatment of deep vein thrombosis and CAT as it significantly shortened both duration of thromboembolic crisis and hospitalization. Safety concerns associated with the bleeding risks are the major barrier to dose optimization of treatments. Here, we used the novel sulfated non-anticoagulant LMWH, referred to as S-NACH, which has a similar activity of LMWH but devoid of systemic anti-factor Xa and IIa activity. The sulfated form has enhanced S-NACH binding to the vascular endothelial cells, release TFPI and potentiate the action of tissue factor pathway inhibitors (TFPI). We investigated the effects of S-NACH on clot kinetics in comparison with LMWH using thrombelstography (TEG) and examined its in vitro and in vivo effects on vascular endothelial release capacity of TFPI. Also we investigate the effects of S-NACH on cancer-associated thrombosis-mediated by human pancreatic cancer cells (SUIT2). Methods: We conducted both in vitro and in vivo experiments using our compound (S-NACH) to test its efficacy on coagulation properties in comparison to LMWH (enoxaparin). We compared the effects on the coagulation time, time to clot initiation and thrombus strength level in vitro, with and without HUVEC endothelial cell line coated into cups used for the TEG analysis. TFPI levels were also estimated in both in vitro and in vivo models at different doses of S-NACH. In addition, the effect of S-NACH on factors Xa and IIa was assessed in mice and rabbits. Also, weused pancreatic cancer cell line (SUIT2) to test the efficiency of blood coagulation in the presence of S-NACH, and Enoxaparin at 1-10 μg. Results: TEG assay have shown that, increasing concentrations of S-NACH (0.1 - 10 ug) did not affect the clotting time (R) and maximum amplitude of the clot formation (MA), while LMWH (enoxaparin) was associated with marked increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. Using HUVEC cell line coated cups; both S-NACH and LMWH were associated with increased clotting time (R) and decreased maximum amplitude of the clot formation (MA). This is due to the ability of HUVEC cell to release of TFPI. In the in vivo study, administration of enoxaparin inhibited both factors Xa and IIa, while administration of S-NACH did not affect the activities of these coagulation factors. S-NACH caused 3 folds greater tissue factor pathway inhibitor (TFPI) release from human endothelial cells and in mice versus enoxaparin. On the other hand, TEG assay shows significantly decreased clotting time (R-value) and increased clot strength in blood from healthy donors when adding pancreatic SUIT2 cells. Data from the TEG assay demonstrated lack of effects of S-NACH or TFPI alone on clot kinetics in human. In contrast, S-NACH (1-10 μg) in the presence of 0.01-0.1 ng TFPI resulted in a dose-dependent suppression of clot strength and prolongation of clotting time (Figure 1). Conclusion: S-NACH is devoid of anti-thrombin binding and inhibition of systemic anti-thrombin-dependent coagulation factors such as factor Xa and factor IIa but has an optimal releasing capacity of vascular endothelial TFPI that is associated with antithrombotic activities without any effect on hemostasis. Also, S-NACH prevents CAT without any effects on hemostasis. Disclosures No relevant conflicts of interest to declare.

Construction and Optimization of Prethrombin-2 Human Genes in E. coli for the Production of Active Thrombin

Prothrombin-2 is a thrombin precursor, which plays an essential role in the conversion of fibrinogen to fibrin during the blood clotting process. In this study, we designed and constructed the human prethrombin-2 (hPT2) gene. hPT2 gene fused with a tag at the n terminal position, which contains the sequence intein followed by chitin binding domain (CBD) that useful for the purification process. The digestion process of hPT2 tag induced by changing its pH/temperature. The hPT2 codon that used in this study designed according to the preference codon of E. coli. hPT2 gene was created using the OPTIMIZER software with the addition of two side restriction at the 5’ and 3’, BamHI and XhoI respectively then were cloned by using the pMAT vector in E. coli. Moreover, hPT2 fragment ligated into an expression vector, pTWIN1, for E. coli. The result from the characterization hPT2 gene in pTWIN1 that using DNA sequencing method shown that the designed hPT2 gene cloned. Thus, the cloned hPT2 gene used as a precursor for PT2 expression in the E. coli host.

A Mathematical Model of Bivalent Binding Suggests Physical Trapping of Thrombin within Fibrin Fibers

Thrombin is an enzyme that plays many important roles in the blood clotting process; it activates platelets, cleaves coagulation proteins within feedback loops, and cleaves fibrinogen into fibrin, which polymerizes into fibers to form a stabilizing gel matrix in and around growing clots. Thrombin also binds to the formed fibrin matrix, but this interaction is not well understood. Thrombin-fibrin binding is often described as two independent, single-step binding events, one high-affinity and one low-affinity. However, kinetic schemes describing these single-step binding events do not explain experimentally-observed residency times of fibrin-bound thrombin. In this work, we study a bivalent, sequential-step binding scheme as an alternative to the high-affinity event and, in addition to the low-affinity one. We developed mathematical models for the single- and sequential-step schemes consisting of reaction-diffusion equations to compare to each other and to experimental data. We then used Bayesian inference, in the form of Markov chain Monte Carlo, to learn model parameter distributions from previously published experimental data. For the model to best fit the data, we made an additional assumption that thrombin was irreversibly sequestered; we hypothesized that this could be due to thrombin becoming physically trapped within fibrin fibers as they formed. We further estimated that ∼30% of thrombin in the experiments to which we compare our model output became physically trapped. The notion of physically trapped thrombin may provide new insights into conflicting observations regarding the speed of fibrinolysis. Finally, we show that our new model can be used to further probe scenarios dealing with thrombin allostery.

Abstract 416: Approach of mRNA Delivery by Using Nanoparticles to Recover Endocardial Notch Signaling

Notch signaling plays a significant role in cardiac development and morphogenesis, especially for cardiac chamber development. Failure of mechanotransduction of endocardial Notch signaling in ventricular chamber causes lack of trabeculation. Advent of nanoparticle (NP) technology offers a therapeutic approach of targeted delivery. Although delivering mRNA is an effective method, it is challenging due to stability and cell defense mechanisms. To overcome the limitations and effectively deliver mRNA to endocardium, we used Poly(lactic-co-glycolic acid) (PLGA) synthesized NPs, which have been used in various drug delivery systems due to its biocompatibility, efficient encapsulation of hydrophilic and hydrophobic materials, specific targeting effects, and lower payload concentration. We hypothesized our PLGA NPs are able to deliver NICD mRNA to target endocardium to overexpress Notch signaling in a Notch inhibited zebrafish animal model. Using double emulsion, we synthesized PLGA NPs at 55–65 kDa and performed in vitro test on HUVECs. 69.8 ± 4.3% of Rhodamine B was successfully loaded into the NPs, and after incubation with HUVECs the signal lasted for 28 days after burst release. Hemolysis and whole blood clotting assays proved that NPs did not interfere with the blood clotting process or lyse blood cells. We characterized mRNA incorporated PLGA nanoparticles and incubated with HUVECs after antibody conjugation. To test in vivo study, we injected gata1a morpholino oligomer (MO) to Tg(fli1:gfp) transgenic line to inhibit hematopoiesis, which reduces wall shear stress (WSS) of ventricular chamber leading lack of trabeculae. At 2 days post fertilization (dpf), NICD mRNA incorporated nanoparticles were injected into the circulatory system of gata1a MO injected zebrafish to rescue trabeculation. At 5 dpf, zebrafish hearts were harvested and analyzed via qRT-PCR to compare Notch related gene expression among control, gata1a MO injected, and rescue group. The NICD mRNA injected group successfully rescued the expression level of Notch components despite a lack of WSS and mechanosensitive Notch signaling via gata1a MO injection. This finding will provide an initial therapeutic approach of NICD mRNA for trabecular related congenital heart disease.

Efek latihan kesamaptaan terhadap kadar D-Dimer pada anggota militer Kodam I Bukit Barisan

Background: Several reports and experiences show that when doing heavy exercise often occur complaints which direct the diagnosis towards Venous Thrombo-Embolism (VTE) in the form of painful and swollen feet. This was due to hypercoagulation and resulted in VTE. The D-dimer level is a measure of clot formation and lysis resulting from the breakdown of fibrinogen and fibrin during fibrinolysis. Therefore this study aimed to see an increase in D-Dimer which is a marker of increased fibrinolysis.Method: This study took a sample of blood from 30 soldiers who were declared healthy. The sample was checked for D-Dimer 1 hour before and after the test. The physical readiness test is a standard for assessing the readiness of soldiers in carrying out tasks. The sample was evaluated whether there was an increase after the test. Examination using Coatron machine with the Immuno Turbidimetric Assay method.Result: 30 people are men with an average age of 19-22 years. D-Dimer showed a significant difference with a mean of 92.83 ± 49.62 SD before the test and after the test found a mean of 127.53 ± 57.75 SD with p = 0.001 (p <0.05).Conclusion: The increase in D-Dimer shows an increase in blood coagulation processes and fibrinolysis and can occur in individuals who often carry out heavy activities. D-Dimer and Dopler ultrasound screening should be carried out on a periodic check before carrying out the physical readiness test.

The Associations between Central Nervous System Diseases and Haemostatic Disorders.

The aim of this review was to examine the relationship between the occurrence of central nervous system (CNS) diseases, the medicines used in their treatment and the blood coagulation process. The paper mainly focuses on the effects of antidepressant and antipsychotic drugs. Special attention has been paid to the influence of drugs on platelets, the vascular endothelium, plasma coagulation and fibrinolysis, regarding coagulation.

Role of charge-reversal in the hemo/immuno-compatibility of polycationic gene delivery systems

As an effective and well-recognized strategy used in many delivery systems, such as polycation gene vectors, charge reversal refers to the alternation of vector surface charge from negative (in blood circulation) to positive (in the targeted tissue) in response to specific stimuli to simultaneously satisfy the requirements of biocompatibility and targeting. Although charge reversal vectors are intended to avoid interactions with blood in their application, no overall or systematic investigation has been carried out to verify the role of charge reversal in the blood compatibility. Herein, we comprehensively mapped the effects of a typical charge-reversible polycation gene vector based on pH-responsive 2,3-dimethylmaleic anhydride (DMMA)-modified polyethylenimine (PEI)/pDNA complex in terms of blood components, coagulation function, and immune response as compared to conventional PEGylated modification. The in vitro and in vivo results displayed that charge-reversal modification significantly improves the PEI/pDNA-induced abnormal effect on vascular endothelial cells, platelet activation, clotting factor activity, fibrinogen polymerization, blood coagulation process, and pro-inflammatory cytokine expression. Unexpectedly, (PEI/pDNA)-DMMA induced the cytoskeleton impairment-mediated erythrocyte morphological alternation and complement activation even more than PEI/pDNA. Further, transcriptome sequencing demonstrated that the overexpression of pro-inflammatory cytokines was correlated with vector-induced differentially expressed gene number and mediated by inflammation-related signaling pathways (MAPK, NF-κB, Toll-like receptor, and JAK-STAT) activation. By comparison, charge-reversal modification improved the hemocompatibility to a greater extent than dose PEGylation except for erythrocyte rupture. Nevertheless, it is inferior to mPEG modification in terms of immunocompatibility. These findings provide comprehensive insights to understand the molecular mechanisms of the effects of charge reversal on blood components and their function and to provide valuable information for its potential applications from laboratory to clinic. Statement of SignificanceThe seemingly revolutionary charge reversal strategy has been believed to possess stealth character with negative charge eluding interaction with blood components during circulation. However to date, no overall or systematic investigation has been carried out to verify the role of charge-reversal on the blood/immune compatibility, which impede their development from laboratory to bedside. Therefore, we comprehensively mapped the effects of a typical charge-reversible polycationic gene vector on blood components (vascular endothelial cell, platelet, clotting factors, fibrinogen, RBCs and coagulation function) and immune response (complement and pro-inflammatory cytokines) at cellular and molecular level in comparison to PEGylation modification. These findings help to elucidate the molecular mechanisms for the effects of charge-reversal on blood components and functions, and provide valuable information for the possible application in clinical settings.

The In Vitro Effects of Pentamidine Isethionate on Coagulation and Fibrinolysis.

Pentamidine is bis-oxybenzamidine-based antiprotozoal drug. The parenteral use of pentamidine appears to affect the processes of blood coagulation and/or fibrinolysis resulting in rare but potentially life-threatening blood clot formation. Pentamidine was also found to cause disseminated intravascular coagulation syndrome. To investigate the potential underlying molecular mechanism(s) of pentamidine’s effects on coagulation and fibrinolysis, we studied its effects on clotting times in normal and deficient human plasmas. Using normal plasma, pentamidine isethionate doubled the activated partial thromboplastin time at 27.5 µM, doubled the prothrombin time at 45.7 µM, and weakly doubled the thrombin time at 158.17 µM. Using plasmas deficient of factors VIIa, IXa, XIa, or XIIa, the concentrations to double the activated partial thromboplastin time were similar to that obtained using normal plasma. Pentamidine also inhibited plasmin-mediated clot lysis with half-maximal inhibitory concentration (IC50) value of ~3.6 μM. Chromogenic substrate hydrolysis assays indicated that pentamidine inhibits factor Xa and plasmin with IC50 values of 10.4 µM and 8.4 µM, respectively. Interestingly, it did not significantly inhibit thrombin, factor XIa, factor XIIIa, neutrophil elastase, or chymotrypsin at the highest concentrations tested. Michaelis-Menten kinetics and molecular modeling studies revealed that pentamidine inhibits factor Xa and plasmin in a competitive fashion. Overall, this study provides quantitative mechanistic insights into the in vitro effects of pentamidine isethionate on coagulation and fibrinolysis via the disruption of the proteolytic activity of factor Xa and plasmin.

PF798 ANTITHROMBOTIC EFFECT OF SULFATED NON‐ANTICOAGULANT HEPARIN WITHOUT COMPROMIZING HEMOSTASIS

Background:The pathophysiology of thrombosis comprises a complex interplay of factors associated with vascular endothelial activation, platelet activation and adhesion. Blood coagulation process initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. Although heparin can be administered therapeutically to prevent and treat thrombosis, the effects of unfractionated heparin are more unpredictable, Low molecular weight heparin (LMWH) was recommended for prophylaxis and treatment of deep vein thrombosis as it significantly shortened both duration of thromboembolic crisis and hospitalization by ∼40%. Safety concerns associated with the narrow therapeutic index (bleeding risks) of LMWH are a major barrier to dose escalation/optimization of treatments. Here, we used the novel sulfated non‐anticoagulant LMWH, referred to as S‐NACH, which has a similar activity of LMWH but devoid of systemic anti‐factor Xa and IIa activity. Also, the sulfated form enhanced S‐NACH binding to the vascular endothelial cells and potentiate the action of tissue factor pathway inhibitors (TFPI‐1 and 2).Aims:To determine if S‐NACH exerts antithrombotic activity without compromizing hemostasis (bleeding) side effects that are associated with the clinical use of LMWHs. In the present study we investigated the effects of S‐NACH on clot kinetics in comparison to LMWH using thrombelstography (TAG) and examined its in vitro and in vivo effects on vascular endothelial release capacity of TFPI.Methods:We conducted both in vitro and in vivo experiments using our compound (S‐NACH) to test its efficacy on coagulation properties in comparison to LMWH (enoxaparin). We compared the effects on the coagulation time, time to clot initiation and thrombus strength level in vitro, with and without HUVEC endothelial cell line coated into cups used for the TEG analysis. TFPI‐1 and TFPI‐2 levels were also estimated in both in vitro and in vivo models at different doses of S‐NACH. In addition, the effect of S‐NACH on factors Xa and IIa was assessed in mice and rabbits.Results:TEG assay have shown that, increasing concentrations of S‐NACH (0.1 – 10 ug) did not affect the clotting time (R) and maximum amplitude of the clot formation (MA), while LMWH (enoxaparin) was associated with marked increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) in comparison to control. Using HUVEC cell line coated cups; both S‐NACH and LMWH were associated with increased clotting time (R) and decreased maximum amplitude of the clot formation (MA) (Table 1). This is due to the ability of HUVEC cell to release of TFPI‐1 and TFPI‐2. In the in vivo study, administration of enoxaparin inhibited both factors Xa and IIa, while administration of S‐NACH did not affect the activities of these coagulation factors. However, S‐NACH caused 3 folds greater tissue factor pathway inhibitor (TFPI) release from human endothelial cells and in mice versus enoxaparin (Figure 1).Summary/Conclusion:S‐NACH is devoid of anti‐thrombin binding and inhibition of systemic anti‐thrombin‐dependent coagulation factors such as factor Xa and factor IIa but has an optimal releasing capacity of vascular endothelial TFPI that is associated with antithrombotic activities without any effect on hemostasis.image

Highly Sensitive Detection of Protein Biomarkers Using Graphene Field-Effect Transistor Integrated with a Microfluidic Platform

In this work, we present a highly sensitive biosensing platform for the detection of protein biomarkers by utilizing aptamer-modified graphene field-effect transistors (GFETs) contained within a microfluidic chip. We have chosen thrombin as a model biomarker and its corresponding DNA-based aptamer as a target receptor which is anchored to the graphene surface to modulate the drain current of the GFET. The proposed biosensing platform has the potential to be used in early-stage disease diagnosis and in applications related to lab-on-a-chip-based health monitoring technology. Aptamer-based biosensing offers many advantages including low-cost, chemical and thermal stability, mass producibility, long shelf-life and reusability. Among different target analytes, protein biomarker detection is critical in early screening and diagnostics of diseases. Thrombin is an important protein biomarker which is produced by the body and plays a significant role in blood clotting or coagulation process or regulation of tumor growth [1]. Therefore, detection of thrombin is key to monitoring a number of cardiovascular diseases. Graphene-based FET (GFET) has become a highly promising candidate as next-generation biosensors due to the various inherent advantages it offers such as facile integration with the existing planar technology due to its 2-dimensional structure, label-free sensing, and high sensitivity. However, one of the major challenges with GFET-based biosensing is the lack of reproducibility due to each graphene layer having different physical dimensions and therefore electronic properties. Moreover, since GFET is highly sensitive to the charge disturbances at the FET channel, the sample liquid that is in contact with the graphene surface must be controlled. Therefore, integration of GFET biosensor with a microfluidic device is expected to enhance the sensing performances in terms of sensitivity and reproducibility. In this work, we have implemented a microfluidic chip integrated with GFET device to build a miniaturized platform for aptamer-based detection of a protein biomarker. The GFET devices were fabricated by transferring CVD (chemical vapor deposition) grown graphene onto the microfabricated gold electrodes on the Si/SiO2substrate where the electrodes form the source and the drainas schematically depicted in Figure 1. The FET devices were then sealed with a PDMS block containing a microfluidic channel as shown in Figure 2. The graphene channel was then functionalized with amine reactive 1-pyrene butyric acid N-hydroxysuccinimide ester (PBASE) by delivering the PBASE dissolved in dimethyl formamide through the microfluidic channel. The PBASE was immobilized on graphene surface via the π-π stacking interaction between graphene and the pyrene group of PBASE. The graphene channel was then further functionalized with the aptamers by flowing the amine terminated thrombin-binding aptamer solution through the microfluidic chip. The aptamers were crosslinked with the PBASE on graphene surface via the covalent linkage between the amine-reactive succinimide group of PBASE and the amine group of the aptamers [2]. For the biomarker detection, various concentrations of thrombin solutions were introduced to the GFET device through the microfluidic flow system. For a given concentration of thrombin, the drain current (IDS) vs. gate voltage (VGS) characteristics were measured in order to observe the shift in the Dirac voltage (VDirac). Figure 3 shows the Raman spectra of graphene on Si/SiO2 substrate before and after PBASE functionalization. The additional peak at 1618 cm-1 in Figure 3(a) represents the pyrene group resonance peak due to π – π stacking interaction [3] between the aromatic pyrenyl group of PBASE and the basal plane of graphene, confirming the immobilization of PBASE on graphene. Figure 4 shows the UV-vis spectra of the device sample before and after the aptamer immobilization. The peak at 260 nm indicates the presence of aptamers on the device surface. The resulting measurements are presented in Figure 5. It can be seen that introduction of thrombin causes the Dirac voltage to shift to the right suggesting p-doping of the graphene surface [4]. This is consistent with the cationic nature of thrombin at neutral pH. The corresponding calibration curve is shown in Figure 6. The sensor shows a linear behavior over a wide range of concentrations from 1 nM to 1µM.

Trends and Prospects of Plant Proteases in Therapeutics.

The main function of proteases in any living organism is the cleavage of proteins resulting in the degradation of damaged, misfolded and potentially harmful proteins and therefore providing the cell with amino acids essential for the synthesis of new proteins. Besides this main function, proteases may play an important role as signal molecules and participate in numerous protein cascades to maintain the vital processes of an organism. Plant proteases are no exception to this rule. Moreover, in contrast to humanencoded enzymes, many plant proteases possess exceptional features such as higher stability, unique substrate specificity and a wide pH range for enzymatic activity. These valuable features make plant-derived proteolytic enzymes suitable for many biomedical applications, and furthermore, the plants can serve as factories for protein production. Plant proteases are already applied in the treatment of several pathological conditions in the human organism. Some of the enzymes possess antitumour, antibacterial and antifungal activity. The collagenolytic activity of plant proteases determines important medical applications such as the healing of wounds and burn debridement. Plant proteases may affect blood coagulation processes and can be applied in the treatment of digestive disorders. The present review summarizes recent advances and possible applications for plant proteases in biomedicine, and proposes further development of plant-derived proteolytic enzymes in the biotechnology and pharmaceutical industries.

Bioinspired Shear-Flow-Driven Layer-by-Layer in Situ Self-Assembly.

Layer-by-layer (LbL) assembly is widely applied as a coating technique for the nanoscale control of architecture and related properties. However, its translational applications are limited by the time-consuming and laborious nature of the process. Inspired by the blood-clotting process, herein, we develop a shear-flow-driven LbL (SF-LbL) self-assembly approach that accelerates the adsorption rate of macromolecules by mechanically configuring the polymer chain via a coil-stretch transition, which effectively simplifies and speeds the diffusion-controlled assembly process. The structural characteristics and surface homogeneity of the SF-LbL films are improved, and diverse three-dimensional structures can be achieved. Functional SF-LbL-assembled surfaces for corneal modification are successfully fabricated, and the surface of wounded rat corneas and skin can be directly decorated in situ with SF-LbL nanofilms due to the advantages of this approach. Furthermore, in situ SF-LbL self-assembly has promise as a simple approach for the wound dressing for interventional therapeutics in the clinic, as illustrated by the successful in situ fabrication of drug-free layers consisting of chitosan and heparin on the dorsal skin of diabetic mice to rescue defective wound healing. This bioinspired self-assembly approach is expected to provide a robust and versatile platform with which to explore the surface engineering of nanofilms in science, engineering, and medicine.

In silico identification of mimicking molecule(s) triggering von Willebrand factor in human: a molecular drug target for regulating coagulation pathway

Blood coagulation is a complex and dynamic process wherein the body activates its emergency mechanism to stop bleeding and wound healing via the interactions of prothrombotic and antithrombotic agents. von Willebrand factor (VWF) is a complex glycoprotein and initial component of the hemostasis pathway which serves a multipurpose role in blood coagulation process. There are reports of various plants that contain several bioactive compounds possessing properties of inducing blood coagulation directly or indirectly. In the present study, efforts have been made to identify bioactive compounds that may play a significant role in regulation of the coagulation cascade by accelerating VWF and thus enhance the hemostasis process. An antidiuretic peptide drug, Desmopressin, works on VWF and releases them in circulation. Forty-seven compounds from different plant sources were screened through molecular docking, out of which two compounds, Emodin and Peruvianoside II, showed more binding affinity than the reference drug Desmopressin. Emodin and Peruvianoside II showed binding energies −7.2 and −7.0 kcal/mol, respectively, when docked with VWF, whereas Desmopressin displayed less binding energy (−6.9 kcal/mol). Emodin belongs to anthraquinone from Rumex hastasus and Peruvianoside II belongs to flavanone glycosides from Thevetia peruviana. The mimicking potential of top identified molecules with respect to the drug was confirmed through simulation analysis. Besides, the molecular dynamics simulation (MDS) study (for 20 ns) showed that the Peruvianoside II protein complex was energetically more stable than Emodin protein complex. Based on the results, Peruvianoside II possesses great potential and thus may be considered for development of drugs for hemostasis.

Interfacial rheology and direct imaging reveal domain-templated network formation in phospholipid monolayers penetrated by fibrinogen.

Phospholipids are found throughout the natural world, including the lung surfactant (LS) layer that reduces pulmonary surface tension and enables breathing. Fibrinogen, a protein involved in the blood clotting process, is implicated in LS inactivation and the progression of disorders such as acute respiratory distress syndrome. However, the interaction between fibrinogen and LS at the air-water interface is poorly understood. Through a combined microrheological, confocal and epifluorescence microscopy approach we quantify the interfacial shear response and directly image the morphological evolution when a model LS monolayer is penetrated by fibrinogen. When injected into the subphase beneath a monolayer of the phospholipid dipalmitoylphosphatidylcholine (DPPC, the majority component of LS), fibrinogen preferentially penetrates disordered liquid expanded (LE) regions and accumulates on the boundaries between LE DPPC and liquid condensed (LC) DPPC domains. Thus, fibrinogen is line active. Aggregates grow from the LC domain boundaries, ultimately forming a percolating network. This network stiffens the interface compared to pure DPPC and imparts the penetrated monolayer with a viscoelastic character reminiscent of a weak gel. When the DPPC monolayer is initially compressed beyond LE-LC coexistence, stiffening is significantly more modest and the penetrated monolayer retains a viscous-dominated, DPPC-like character.