Whole Blood Clotting Research Articles

Patients with Waldenström macroglobulinemia have impaired platelet and coagulation function

AbstractClinical features in patients with the B-cell lymphoma, Waldenström macroglobulinemia (WM), include cytopenias, immunoglobulin M (IgM)–mediated hyperviscosity, fatigue, bleeding, and bruising. Therapeutics such as Bruton's tyrosine kinase inhibitors (BTKis) exacerbate bleeding risk. Abnormal hemostasis arising from platelet dysfunction, altered coagulation or vascular impairment have not yet been investigated in patients with WM. Therefore, this study aimed to evaluate hemostatic dysfunction in samples from these patients. Whole blood (WB) samples were collected from 14 patients with WM not receiving therapy, 5 patients receiving BTKis and 15 healthy donors (HDs). Platelet receptor levels and reticulation were measured by flow cytometry, plasma thrombin generation with or without platelets by fluorescence resonance energy transfer assay, WB clotting potential by rotational thromboelastometry, and plasma soluble glycoprotein VI (sGPVI) and serum thrombopoietin (TPO) by enzyme-linked immunosorbent assay. Donor platelet spreading, aggregation, and ability to accelerate thrombin generation in the presence of WM-derived IgM were assessed. WM platelet receptor levels, responses to physiological agonists, and plasma sGPVI were within normal ranges. WM platelets had reduced reticulation (P = .0012) whereas serum TPO levels were increased (P = .0040). WM plasma displayed slower thrombin generation (P = .0080) and WM platelets contributed less to endogenous thrombin potential (ETP; P = .0312). HD plasma or platelets incubated with IgM (50-60 mg/mL) displayed reduced spreading (P = .0002), aggregation (P < .0001), and ETP (P = .0081). Thus, alterations to thrombin potential and WB coagulation were detected in WM samples. WM IgM significantly impaired hemostasis in vitro. Platelet and coagulation properties are disturbed in patients with well-managed WM.

Efficacy and safety of RD2 Ver.02, a whole blood clot therapy, coupled with a minimally invasive procedure in pilonidal sinus: a phase II study.

PNS is caused by an infection in the sacrococcygeal area triggered by hair particle accumulation in skin tunnels, resulting in infection. Surgical options range from simple excision to complex flap constructions. Primary wound healing failure and recurrence rates contribute to the burden of PNS. RD2 Ver.02, a novel autologous whole-blood clot product, demonstrated safety and efficacy in treating complex cutaneous wounds and was investigated for the management of PNS. A Phase II open-label, pilot, single-arm prospective study was conducted from May 2021 to May 2023 (Ethics Committee approval #7952-20). Patients with PNS underwent a minimally invasive trephine procedure under local anesthesia followed by RD2 Ver.02 instillation into the cavity. Primary healing was assessed at 3, 6, and 12 months. Secondary outcomes included the collection of adverse events. Overall, 51 patients participated in the study. At 3 months, 42/51 healed (82.4%), 7/51 (13.7%) were granulating but not completely healed, and 2/51 (3.9%) failed to heal. At 6 and 12 months, 46/51 (90.2%) and 42/51 (82.4%) achieved complete healing, respectively. At 6 months, two PNSs recurred after initial healing and an additional four instances of PNS recurrence observed in 12months, so a total of recurrence in six patients (11.8%). There were five adverse events (AEs) with no severe adverse events. RD2 Ver.02 is a safe and effective treatment of PNS when coupled with a minimally invasive trephine PNS procedure. Further comparative studies are needed to fully assess the role of this novel therapy for PNS.

The fibrinolytic potential of Bacillus amyloliquefaciens isolates from salt-fermented shrimp paste terasi

Abstract. Pinontoan R, City S, Widjaja AN, Purnomo JS, Dikson. 2024. The fibrinolytic potential of Bacillus amyloliquefaciens isolates from salt-fermented shrimp paste terasi. Biodiversitas 25: 3193-3199. Thrombosis, a major pathology in Cardiovascular Diseases (CVDs), significantly contributes to global mortality. Although medicinal prevention and management of recurrent thromboses do exist, alternative means using natural sources are actively sought because of their lower costs, better compatibility, and lower risks of side effects. Due to their beneficial microorganisms, fermented foods offer a potential thrombolytic source for managing CVDs. In this study, we aimed to isolate and identify bacteria with thrombolytic activity from fermented shrimp paste terasi. Potential protease-producing bacteria from terasi were determined via cell and colony morphology, biochemical properties, and 16S rRNA sequence analyses. Subsequently, the thrombolytic and fibrinolytic activities of the bacteria were assessed by performing whole-blood clot tests and fibrin degradation assays; two protease-producing bacteria, designated as TJU5 and TMAD4 isolates were identified as Bacillus amyloliquefaciens. The isolates demonstrated thrombolytic activity by significantly reducing whole-blood clot mass after 2 h of incubation. The thrombolytic mechanism involves fibrinolysis indicated by the rapid degradation of A?, B?, and ? fibrin chains observed within 1 min of incubation. These findings highlight the beneficial bacteria from fermented shrimp paste terasi, identified as B. amyloliquefaciens TJU5 and TMAD4, with high thrombolytic and fibrinolytic activities, underscoring their potential role in bolstering cardiovascular health.

Wound healing and coagulant activity of crude extract metabolites from fungal endophytes

Bleeding from a wound as a result of physical injury is a life-threatening condition. In pursuing new drug structures, the effect of crude fungal extracts fungal isolated from Jatropha multifida on wound healing and coagulation of mouse whole blood was investigated. Jatropha multifida leaves were sterilized, cut into small segments, and then incubated in Potato Dextrose Agar for seven days. Four isolates were purified and their morphologies were characterized. Identification of isolates was confirmed by a molecular protocol. Two crude extracts from Phlebiopsis gigantea (OK021602) and Phyllosticta sp (OK021603), which exhibited higher phytochemicals composition, were selected and evaluated using wound excision and coagulation of mouse whole blood, by administering 30 µg/mL, 50 µg/mL and 70 µg/mL crude extracts respectively. The percentage of wound healing in mice was higher (p&amp;lt;0.05) for the crude extracts of Phlebiopsis gigantea (OK021602) as compared to that of Phyllosticta sp (OK021603). The highest percentages of wound contraction were 99% at 70 µg/mL, and 53% at 70 µg/mL for Phlebiopsis gigantea (OK021602) and Phyllosticta sp (OK021603), respectively as compared to the control group which had 42% wound contraction at day 15 post-treatment. The results of the present study clearly indicate that Jatropha multifida leaves harbor endophytic fungi that produce pharmacologically important bioactive secondary metabolites with wound and hemostatic effects; therefore, further exploration is inevitable, particularly for the purification and identification of specific chemical structures of bioactive compounds.

Material strengths of shear-induced platelet aggregation clots and coagulation clots

Arterial occlusion by thrombosis is the immediate cause of some strokes, heart attacks, and peripheral artery disease. Most prior studies assume that coagulation creates the thrombus. However, a contradiction arises as whole blood (WB) clots from coagulation are too weak to stop arterial blood pressures (> 150 mmHg). We measure the material mechanical properties of elasticity and ultimate strength for Shear-Induced Platelet Aggregation (SIPA) type clots, that form under stenotic arterial hemodynamics in comparison with coagulation clots. The ultimate strength of SIPA clots averaged 4.6 ± 1.3 kPa, while WB coagulation clots had a strength of 0.63 ± 0.3 kPa (p < 0.05). The elastic modulus of SIPA clots was 3.8 ± 1.5 kPa at 1 Hz and 0.5 mm displacement, or 2.8 times higher than WB coagulation clots (1.3 ± 1.2 kPa, p < 0.0001). This study shows that the SIPA thrombi, formed quickly under high shear hemodynamics, is seven-fold stronger and three-fold stiffer compared to WB coagulation clots. A force balance calculation shows a SIPA clot has the strength to resist arterial pressure with a short length of less than 2 mm, consistent with coronary pathology.

Clinical Laboratory Investigations and Antivenom Administration after Malayan Pit Viper (Calloselasma rhodostoma) Envenoming: A Retrospective Study from Southernmost Thailand.

The Malayan pit viper (MPV: Calloselasma rhodostoma) is a medically important venomous snake causing numerous envenomations in Thailand. Administration of specific snake antivenom is the only effective treatment for MPV-envenomed patients. However, inappropriate administration or misuse of snake antivenom is problematic in some remote areas of tropical countries where the snakebite envenoming rate is notable. Currently, the indications for administration of MPV antivenom are focused mainly on hematological factors. These include 1) venous clotting time > 20 min, 2) unclotted 20-minute whole-blood clotting time, 3) international normalized ratio > 1.2, 4) platelet count < 50 × 103/μL, 5) systemic bleeding, and 6) impending compartment syndrome. We aimed to determine the association between laboratory data and antivenom administration in MPV-envenomed patients. A retrospective study of data from 2016 to 2021 in Narathiwat Province, the southernmost province in Thailand, was conducted. A total of 838 MPV-bitten patients were included in this study. Local effects and systemic effects were observed in 58.8% and 27.7% of patients, respectively. Coagulopathies, which range from abnormal blood clotting to systemic bleeding, represented the majority of systemic effects. Acute kidney injury developed in 2.5% of patients. In this study, 57.3% of patients were considered appropriate antivenom recipients. Interestingly, the present study revealed that local bleeding and mild to moderate thrombocytopenia became the independent factors for inappropriate use of MPV antivenom. Reeducation and supervision regarding the rational use of snake antivenom are needed to minimize the misuse of antivenom.

Preparation and Performance Study of a Novel Antibacterial Hemostatic Chitosan Sponge

To create a novel chitosan antibacterial hemostatic sponge (NCAHS) and to evaluate its material and biological properties. Chitosan, a polysaccharide, was used as the sponge substrate and different proportions of sodium tripolyphosphate (STPP), glycerol, and phenol sulfonyl ethylamine were added to prepare the sponges through the freeze-drying method. The whole-blood coagulation index (BCI) was used as the screening criterion to determine the optimal concentrations of chitosan and the other additives and the hemostatic sponges were prepared accordingly. Zein/calcium carbonate (Zein/CaCO3) composite microspheres loaded with ciprofloxacin hydrochloride were prepared and added to the hemostatic sponges to obtain NCAHS. Scanning electron microscope was used to observe the microscopic morphology and porosity of the NCAHS. The water absorption rate, in vitro antibacterial susceptibility rate against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), in vitro coagulation performance, and hemocompatibility of NCAHS were examined. The coagulation performance of NCAHS was evaluated by using rabbit liver injury and rabbit auricular artery hemorrhageear models and commercial hemostatic sponge (CHS) was used as a control. The in vivo biocompatibility, including such aspects as cytotoxicity, skin irritation in animals, and acute in vivo toxicity, of the NCAHS extracts was examined by using as a reference the national standards for biological evaluation of medical devices. The NCAHS prepared with 1.5% chitosan (W/V), 0.01% STPP (W/V), 0% glycerol (V/V), 0.15% phenol-sulfonyl-ethylamine (V/V), Zein and CaCO3 at the mixing ratio of 5∶1 (W/W), Zein at the final mass concentration of 2.5 g/L, and ethanol at the final concentration of 17.5% (V/V) were fine and homogeneous, possessing a honeycomb-like porous structure with a pore size of about 200 μm. The NCAHS thus prepared had the lowest BCI value. The water absorption ([2362.16±201.15] % vs. [1102.56±91.79]%) and in vitro coagulation performance (31.338% vs. 1.591%) of NCAHS were significantly better than those of CHS (P<0.01). Tests with the in vivo auricular artery hemorrhage model ([36.00±13.42] s vs. [80.00±17.32] s) and rabbit liver bleeding model ([30.00±0] s vs. [70.00±17.32] s) showed that the hemostasis time of NCAHS was significantly shorter than that of CHS (P<0.01). NCAHS had significant inhibitory ability against S. aureus and E. coli. In addition, NCAHS showed good in vitro and in vivo biocompatibility. NCAHS is a composite sponge that shows excellent antimicrobial properties, hemostatic effect, and biocompatibility. Therefore, its extensive application in clinical settings is warranted.

Diversity of Plasmatic Thrombin Pools Regulate Thrombin Generation and Blood Clotting: Interference By a Novel Nanobody

Background: Thrombin has a multitude of roles in the initiation and propagation of (anti)coagulation, in platelet activation and fibrin clot formation. The assessment of thrombin generation (TG) and thrombin-dynamics, combined with multi-omics data, has provided in-depth understanding of the plasma factors contributing to the (in)activation of thrombin in triggered plasma and whole-blood. By default, the generated thrombin is considered to act as a single pool. Here, we present evidence for at least two major proteolytically active thrombin pools with non-overlapping functions in blood clotting. Methods: A novel Syn-Nb-AF106 anti-fibrin nanobody (Nb106) was characterized, targeting a species-conserved thrombin-binding site exposed by fibrinopeptide A cleavage from fibrinogen (Sun et al, this meeting). Thrombin generation was assessed by calibrated automated thrombography using various triggers with control (multi-donor) and (reconstituted) coagulation factor-deficient plasmas, platelet-rich plasma or whole blood. Fibrin clot formation was evaluated by scanning electron microscopy (SEM). Clot retraction was macroscopically assessed. Results: In tissue factor-triggered thrombin generation,we discovered that Nb106 dose-dependently reduced the thrombin peak level and endogenous thrombin potential (ETP) to 54-62%, without affecting kinetic thrombin generation parameters. We observed a similar reduction in platelet-free plasma, platelet-rich plasma and whole blood. Biochemically, Nb106 displaced thrombin from a binding site on cleaved fibrinogen (Sun et al., abstract). Nb106 inhibited tissue factor-induced thrombin generation, using plasma deficient in factor IX, XI or XII, but not using plasmas deficient in fibrinogen or antithrombin. Reconstitution experiments showed that the inhibition depended on the fibrinogen concentration. A-specific control nanobodies were without effect. Together, the fibrin(ogen)-dependent and thrombin-inhibiting effect indicated that Nb106 operates by releasing proteolytically-active thrombin from fibrin to allow inactivation by antithrombin. Time-dependent, late spiking experiments revealed that Nb106 reduced the generation of thrombin only when added at 0-15 minutes after trigger, but no longer after 30 minutes, suggesting that the inhibitory capacity was due to a shortened thrombin binding to fibrin rather than to a continued protection towards antithrombin. In plasmas from a cohort of 64 healthy subjects, application of Nb106 showed a consistent reducing effect on the parameters peak and ETP, with a moderate positive correlation with the fibrinogen level. Nb106 suppressed the thrombin generation amplitude, but not the kinetics in the presence of a panel of direct oral anticoagulants (DOAC). Strikingly, brightfield microscopy and SEM indicated that Nb106, but not control nanobodies, in triggered plasma, platelet-rich plasma and whole-blood, fully abrogated the formation of elongating fibrin fibers. In agreement with this, it strongly suppressed whole-blood clot retraction. Conclusion: Collectivity, these data point to the existence of two major pools of proteolytically active thrombin, both of which contributing to thrombin generation, that are formed upon plasma and blood coagulation. One pool of soluble thrombin steers the proteolytic coagulation cascade, while another pool is bound to growing fibrin fibers. The latter pool still cleaves the conventional AMC thrombin substrate, is temporarily protected against antithrombin, and is required for fibrin fiber extension and blood clotting and clot retraction. The Nb106, replacing thrombin from its binding site on fibrin, selectively annuls the second pool, thereby blocking the clotting process. The replacement ensures thrombin inactivation by antithrombin. Perspective: The novel anti-fibrin nanobody Syn-Nb-AF106 or a derivative has the potential to act as an alternative anticoagulant in thrombotic diseases with pathological fibrin formation.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial

AbstractData from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSβ0thal, HbSβ+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

Polyester urethane urea (PEUU) functionalization for enhanced anti-thrombotic performance: advancing regenerative cardiovascular devices through innovative surface modifications.

Introduction: Thrombogenesis, a major cause of implantable cardiovascular device failure, can be addressed through the use of biodegradable polymers modified with anticoagulating moieties. This study introduces a novel polyester urethane urea (PEUU) functionalized with various anti-platelet deposition molecules for enhanced antiplatelet performance in regenerative cardiovascular devices. Methods: PEUU, synthesized from poly-caprolactone, 1,4-diisocyanatobutane, and putrescine, was chemically oxidized to introduce carboxyl groups, creating PEUU-COOH. This polymer was functionalized in situ with polyethyleneimine, 4-arm polyethylene glycol, seleno-L-cystine, heparin sodium, and fondaparinux. Functionalization was confirmed using Fourier-transformed infrared spectroscopy and X-ray photoelectron spectroscopy. Bio-compatibility and hemocompatibility were validated through metabolic activity and hemolysis assays. The anti-thrombotic activity was assessed using platelet aggregation, lactate dehydrogenase activation assays, and scanning electron microscopy surface imaging. The whole-blood clotting time quantification assay was employed to evaluate anticoagulation properties. Results: Results demonstrated high biocompatibility and hemocompatibility, with the most potent anti-thrombotic activity observed on pegylated surfaces. However, seleno-L-cystine and fondaparinux exhibited no anti-platelet activity. Discussion: The findings highlight the importance of balancing various factors and addressing challenges associated with different approaches when developing innovative surface modifications for cardiovascular devices.

Blood Clotting Dissolution in the Presence of a Magnetic Field and Preliminary Study with MG63 Osteoblast-like Cells-Further Developments for Guided Bone Regeneration?

The influence of a magnetic field on the activation of bone cells and remodelling of alveolar bone is known to incite bone regeneration. Guided Bone Regeneration (GBR) aims to develop biomimetic scaffolds to allow for the functioning of the barrier and the precise succession of wound healing steps, including haemostasis. The effect of a magnetic field on blood clot dissolution has not been studied yet. We conducted a methodological study on the clot stability in the presence of a static magnetic field (SMF). Preformed whole blood (WB) clots were treated with either a broad proteolytic enzyme (trypsin) or a specific fibrinolytic agent, i.e., tissue-type plasminogen activator (t-PA). MG63 osteoblast-like cells were added to preformed WB clots to assess cell proliferation. After having experienced a number of clotting and dissolution protocols, we obtained clot stability exerted by SMF when tissue factor (for clotting) and t-PA + plasminogen (for fibrinolysis) were used. WB clots allowed osteoblast-like cells to survive and proliferate, however no obvious effects of the magnetic field were noted. Paramagnetic properties of erythrocytes may have influenced the reduction in clot dissolution. Future studies are warranted to fully exploit the combination of magnetic forces, WB clot and cells in GBR applied to orthodontics and prosthodontics.

High-resolution characterization of the coagulation and drying processes of whole blood based on optical coherence tomography

Introduction: Escaping whole blood exhibits biochemical and physical coupled mechanisms such as coagulation and drying. However, there is no method for simultaneously monitoring the coagulation and drying procedure.Methods: In this study, a new method based on optical coherence tomography (OCT) combined with speckle variance and thickness is presented for simultaneously capturing spatially high-resolved characteristics of coagulation and drying of whole blood during the procedure. Deep learning based on a convolutional neural network (CNN) is employed for collecting OCT images with a resolution of micron order and quantitatively obtaining pixel-wise information of whole blood.Results and discussion: Then, the pixel-wise thickness map provides high-resolved temporal–spatial dynamics of whole blood during the drying procedure, and the corresponding speckle variance can uncover information of whole blood coagulation. The results demonstrate that coagulation and drying of whole blood have spatially inhomogeneous features. This method could provide the potential for revealing the coupling mechanism between coagulation and drying.

Tyrame [N-(3-hydroxy-1:3:5(10)-estratrien-17β-yl)-4-hydroxy-phenethylamine], antithrombotic aminoestrogen that decreases microvesicle formation.

Estrogens that are used as contraceptives or in replacement therapy are associated with an increase in the risk for developing thrombosis, mainly during the first year of treatment and in women with associated risk factors. To synthesize, characterize and identify the anticoagulant, antiplatelet aggregation and microvesicle-reducing effect of the new aminoestrogen Tyrame. CD1 strain mice were used, which had Tyrame (0, 1 and 2 mg/100 g) subcutaneously administered. At 24 h, a blood sample was obtained to determine whole-blood clotting time, microvesicles concentration and inhibitory effect on platelet aggregation. Blood clotting time increased up to 1.5 times in comparison with the control. Platelet aggregation inhibition had different magnitude depending on the agonist agent employed, and was complete with collagen. Both effects had a dose-dependent relationship. The microvesicles decreased up to six times with respect to the control. Tyrame reduces platelet aggregation and microvesicle formation, which emphasizes its potential therapeutic utility as an estrogen free of thrombotic effects.

Evaluation of a Viscoelastic Coagulation Monitoring System (VCM Vet®) and Its Correlation with Thromboelastometry (ROTEM®) in Diseased and Healthy Dogs.

Thromboelastometry provides a real-time assessment of global coagulation in whole blood. A novel bed-side viscoelastic coagulation monitor (VCM) has been developed for use in small animals. The aims of the study were to determine inter-device agreement of two VCM devices, to evaluate the correlation between VCM and rotational thromboelastometry as a reference method (ROTEM), and to determine the sensitivity and specificity of VCM to diagnose hypo-, normo-, and hypercoagulability. ROTEM (extrinsic and intrinsic activation) analysis was performed using citrated blood and VCM analysis using native blood. Twenty healthy and forty diseased dogs with and without coagulopathies were enrolled. The VCM inter-device agreement was moderate to strong for most of the parameters, depending on the grading scale. Correlation between VCM and ROTEM was moderate to strong for parameters of clotting time and clot strength. The VCM most likely detects true hypocoagulability and reliably rules out hypocoagulability. The VCM has a high sensitivity in diagnosing normocoagulability, but incorrectly classified dogs with abnormal coagulation as normocoagulable. The VCM was not able to detect hypercoagulability. ROTEM and VCM cannot be used interchangeably.

A Pilot Study Evaluating the Ex Vivo Effects of Varying Factor VIII Concentration on Clot Kinetics and Architecture in Patients With Haemophilia A

Background Haemophilia A (HA) is a bleeding disorder, due to a deficiency in factor VIII (FVIII). These patients are unable to produce a stable fibrin clot in the propagation phase of coagulation as they do not generate sufficient thrombin. The primary treatment for HA in South Africa remains replacement therapy with standard half-life FVIII clotting factor concentrate, aimed at reducing bleeding episodes. Objectives To evaluate the effect of varying concentrations of FVIII on whole blood (WB) clot architecture and kinetics during clot formation in patients with severe HA. Design A cross-sectional study where blood from 20 patients with HA was exposed to FVIII ex vivo and compared to a control group of 20 healthy individuals. Methods Scanning electron microscopy (SEM) was used to study WB clot architecture and thromboelastography® (TEG®) was used to quantify WB clot kinetics. Results Scanning electron microscopy studies revealed that patients with HA have sparse, disorganized fibrin networks with limited crosslinking and red blood cells (RBCs) stacked in rouleaux formation. Haemophilia A blood spiked to a 10 to 15 IU/dL FVIII concentration showed improvements in the organisation of the fibrin network with some altered RBCs. In addition, blood spiked to a 30 to 35 IU/dL FVIII concentration showed an increase in fibrin formation with normal RBCs. Thromboelastography® showed that patients with HA had an increased clot initiation time and decreased clot strength. When spiked to a 10 to 15 IU/dL FVIII concentration the clot kinetic profile showed normalization. However, an increase in FVIII concentration higher than 30 IU/dL showed altered clot architecture and kinetics. Conclusion Based on the current study, FVIII levels at 10 to 15 IU/dL improved clot kinetics but did not normalize the architecture. It may be sufficient for prevention of haemorrhages. Factor VIII levels at 30 to 35 IU/dL resulted in rapid but weaker clot formation. However, at this concentration the clot architecture was normalised which is important for haemostasis. Here it was also evident that the findings of these two modalities (TEG® and SEM) should not be separated but interpreted in conjunction with each other.

Gelatin/calcium chloride electrospun nanofibers for rapid hemostasis.

Blood coagulation is the body's main defense to bleeding caused by trauma and is divided into endogenous and exogenous pathways. Calcium ions play a very important role in the process of blood coagulation, as the ions activate the many enzymes that are required for coagulation. In this paper, gelatin hemostatic membranes containing calcium ions were prepared by electrospinning. The fibers were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The biocompatibility and coagulation processes using the calcium ion-containing gelatin fibrous membranes were evaluated in vitro with dynamic whole-blood coagulation tests, hemolysis tests, coagulation time tests, and platelet adhesion tests. It was demonstrated that the calcium ion-containing gelatin membranes had lower hemolysis rates and shorter clotting times than commercially available hemostatic sponges and hemostatic gauzes. In vivo hemostasis experiments were also conducted on the tail vein and liver of mice. Animal experiments demonstrated that the incorporation of calcium ions into the electrospun gelatin membranes promoted platelet aggregation, ensured adhesion of the electrospun membrane to the wound and reduced the bleeding volume and hemostasis time. The composite calcium ion-gelatin electrospun membranes exhibited good in vivo and in vitro hemostatic abilities and accelerated blood clotting by stimulating the coagulation pathway to promote platelet aggregation at the wounds and the formation of mature blood clots for a new approach for acute trauma treatment.

Synthesis of cross-linked polymer based on purified Sterculia foetida L. gum as a potential hemostatic agent

A method for purifying the polysaccharides from Sterculia foetida L. gum was developed. The effects of purifying conditions were also studied. Results have shown that the conditions of pH 0.5, temperature of 80°C, and 2.5-h reaction duration gave the best polysaccharides yield. The mineral content and the molecular weight of the obtained polysaccharides were measured for characterization. The cross-linking of the polysaccharides with citric acid in order to achieve a desired polymer was performed successfully. The obtained polymers under different conditions were evaluated for the potential application as a hemostatic agent, including tests of swelling behavior in different fluids, whole-blood clotting time and enzymatic bio-degradation. Results have shown that the citric acid/polysaccharides ratio of 0.01 produced a comparative yield of polymer flakes with increased swelling properties, as well as shortened in vitro blood clotting time and gradual biodegradability in three experimental days. Therefore, the citric acid/polysaccharides ratio of 0.01 was selected for the subsequent experiment to obtain the polymer after 1.5 h of purification, which gave the best swelling properties, 20 times the initial weight, reduced the whole-blood clotting time by 50% and showed a rapid bio-degradation. The results obtained from this study provide essential knowledge on the research of the use of S. foetida gum polysaccharides as a passive hemostatic agent, thereby extending the potential pharmaceutical applications of natural polysaccharides in Vietnam.

SOIL BACTERIAL ISOLATES SHOWING PROMISING FIBRINOLYTIC ACTIVITY

Directly acting fibrinolytic enzyme is found to be superior therapy for treatment of cardiovascular diseases. Hence, thereis a need of a novel and highly potent fibrinolytic agent which can directly degrade fibrin clot in lesser time withimproved specificity and overcome shortcomings of haemorrhage. Amongst various sources, microbial fibrinolyticenzymes have attracted attention of research community so the present study aimed to isolate fibrinolytic enzymeproducingbacteria from different soil samples. Caseinolytic activity and clot lyses activity of the crude enzyme producedby these isolates were determined. All together six caseinolytic organisms were isolated, 3 of them produced fibrinolyticprotease which would lyse fibrin and whole blood clot. Caseinolytic activity in the crude enzyme preparation fromColony C₄ was detected to be 78.2 U/ml and was able to lyse 85.54% of whole blood clot in 90 mins. Colony 4 may beof potential interest for biotechnological use as it produces fibrinolytic proteases that degraded fibrin clot and the wholeblood clot, to the maximum.

Proactive Hemocompatibility Platform Initiated by PAMAM Dendrimer Adapting to Key Components in Coagulation System.

Surface modification manipulates the application performance of materials, and thrombosis caused by material contact is a key risk factor of biomaterials failure in blood-contacting/implanting devices. Therefore, building a safe and effective hemocompatibility platform is still urgent. Owing to the unique properties of polyamidoamine (PAMAM) dendrimers, in this study, modified surfaces with varying dendrimer densities were interacted with elements maintaining blood homeostasis. These included the plasma proteins bovine serum albumin and fibrinogen, cells in blood (platelets and erythrocyte), as well as endothelial cells (ECs), and the objective was to evaluate the blood compatibility of the chosen materials. Whole blood test and dynamic blood circulation experiment by the arteriovenous shunt mode of rabbit were also conducted, based on the complexity and fluidity of blood. The PAMAM-modified substrates, particularly that with a high density of PAMAM (N1.0), adsorbed proteins with lessened fibrinogen adsorption, reduced platelet activation and aggregation, and suppressed clotting in whole blood and dynamic blood testing. Furthermore, the designed PAMAM dendrimer densities were safe and showed negligible erythrocyte lysis. Concurrently, PAMAM modification could maintain EC growth and did not trigger the release of procoagulant factors. These results suggest that the PAMAM-modified materials are compatible for maintaining blood homeostasis. Thus, PAMAM dendrimers can work as excellent surface modifiers for constructing a hemocompatibility platform and even a primer layer for desired functional design, promoting the service performance of blood-contacting devices.

Potential of a Composite Conduit with Bacterial Nanocellulose and Fish Gelatin for Application as Small-Diameter Artificial Blood Vessel.

Bacterial nanocellulose (BNC) has received great attention for application as an artificial blood vessel material. However, many results showed that pristine BNC could not perfectly meet all the demands of blood vessels, especially for rapid endothelialization. In order to improve the properties of small-caliber vessels, different concentrations of fish gelatin (Gel) were deposited into the 3D network tubes and their properties were explored. The BNC/Gel composite tubes were treated with glutaraldehyde to crosslink BNC and fish gelatin. Compared with pristine BNC tubes, the BNC/Gel tubes had a certain improvement in mechanical properties. In vitro cell culture demonstrated that the human endothelial cells (HUVECs) and human smooth muscle cells (HSMCs) planted on the internal walls of BNC/Gel tubes showed better adhesion, higher proliferation and differentiation potential, and a better anticoagulation property, as compared to the cells cultured on pristine BNC tubes. Whole-blood coagulation experiments showed that the BNC/Gel tube had better properties than the BNC tube, and the hemolysis rate of all samples was less than 1.0%, satisfying the international standards for medical materials. An increase in the content of fish gelatin also increased the mechanical properties and the biocompatibility of small-caliber vessels. Considering the properties of BNC/Gel tubes, 1.0 wt/v% was selected as the most appropriate concentration of fish gelatin for a composite.