Clot Rate Research Articles

Retrospective analysis comparing complication rates of centrifuge vs membrane-based therapeutic plasma exchange in the pediatric population.

There are two conventional modalities used to perform therapeutic plasma exchange (TPE): centrifuge TPE (cTPE) or membrane TPE (mTPE). There is limited data on complications with mTPE. We sought to better understand the patient and machine complications of mTPE compared to cTPE. We hypothesize that our protocol for mTPE using heparin anticoagulation is well-tolerated. In this retrospective cohort study of children <21 years of age, we evaluated differences in patient and machine characteristics and complications between cTPE (with citrate anticoagulation) vs mTPE (with heparin anticoagulation). Of the 105 patients who met inclusion/exclusion criteria, 63 received cTPE and 42 mTPE via Prismaflex. Those who used mTPE were younger (4.8 ± 2.8 years vs 15.2 ± 3.7 years, P=.0001) and weighed less (19.5 ± 10.6 vs 71.7 ± 28.5 kg, P=.0001). There were no significant differences in patient-related complications or indications for TPE between the two modalities. Of the 1031 therapies performed,1003 therapies were analyzed (646 using cTPE and 357 using mTPE) due to exclusion criteria. No significant difference in patient complications were detected between groups. Machine-related complications were infrequent in both approaches. More circuits clotted during mTPE than during cTPE (6.7% [24/357] vs 0% [0/646]; P < 0.001). Although we use mTPE in smaller children, we showed low rates of complications that were not statistically different from cTPE performed in older children. While the overall rate of circuit clotting using mTPE was low, it occurred more commonly than with cTPE.

Development of Cellulose Nanofibril/Casein-Based 3D Composite Hemostasis Scaffold for Potential Wound-Healing Application.

Excessive bleeding in traumatic hemorrhage is the primary concern for natural wound healing and the main reason for trauma deaths. The three-dimensional (3D) bioprinting of bioinks offers the desired structural complexity vital for hemostasis activity and targeted cell proliferation in rapid and controlled wound healing. However, it is challenging to develop suitable bioinks to fabricate specific 3D scaffolds desirable in wound healing. In this work, a 3D composite scaffold is designed using bioprinting technology and synergistic hemostasis mechanisms of cellulose nanofibrils (TCNFs), chitosan, and casein to control blood loss in traumatic hemorrhage. Bioinks that consist of casein bioconjugated TCNF (with a casein content of 104.5 ± 34.1 mg/g) using the carbodiimide cross-linker chemistry were subjected to bioprinting for customizable 3D scaffold fabrication. Further, the 3D composite scaffolds were in situ cross-linked using a green ionic complexation approach. The covalent conjugation among TCNF, casein, and chitosan was confirmed by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and X-ray diffraction (XRD) studies. The in vitro hemostasis activity of the 3D composite scaffold was analyzed by a human thrombin-antithrombin (TAT) assay and adsorption of red blood cells (RBCs) and platelets. The 3D composite scaffold had a better swelling behavior and a faster whole blood clotting rate at each time point than the 3D TCNF scaffold and commercial cellulose-based dressings. The TAT assay demonstrated that the 3D composite scaffold could form a higher content of thrombin (663.29 pg/mL) and stable blood clot compared to a cellulosic pad (580.35 pg/mL), 3D TCNF (457.78 pg/mL), and cellulosic gauze (328.92 pg/mL), which are essential for faster blood coagulation. In addition, the 3D composite scaffold had a lower blood clotting index (23.34%) than the 3D TCNF scaffold (41.93%), suggesting higher efficiencies for RBC entrapping to induce blood clotting. The in vivo cytocompatibility was evaluated by a 3D cell culture study, and results showed that the 3D composite scaffold could promote growth and proliferation of NIH 3T3 fibroblast cells, which is vital for wound healing. Cellulase-based in vitro deconstruction of the 3D composite scaffold showed significant weight loss (80 ± 5%) compared to the lysozyme hydrolysis (22 ± 5%) after 28 days of incubation, suggesting the biodegradation potential of the composite scaffold. In conclusion, this study proposes efficient prospects to develop a 3D composite scaffold from bioprinting of TCNF-based bioinks that can accelerate blood clotting and wound healing, suggesting its potential application in reducing blood loss during traumatic hemorrhage.

Small-Volume Noncontact Assessment of Blood Coagulation Via Acoustic Tweezing Coagulometry

Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as “global coagulation analysis”, mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume.Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph.Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group.Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia.Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. [Display omitted] DisclosuresKasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Anticoagulant profile of subcutaneous enoxaparin in healthy dogs.

Our study objective was to identify a subcutaneous enoxaparin dosage that provided a consistent anticoagulant intensity in dogs. Our hypotheses were that a dose of 0.8mg/kg would provide inconsistent anticoagulation, a higher dose would provide consistent anticoagulation over a greater duration of time, and viscoelastometry would effectively monitor the anticoagulant status. Six healthy dogs received two subcutaneous enoxaparin doses (0.8 and 2mg/kg) for anti-Xa activity determinations and pharmacokinetic modeling. Based on calculations derived from these results, 1.3mg/kg, SC, q8h was administered for seven doses. Target ranges for anticoagulant intensity were defined as anti-Xa activity of 0.5-1U/ml, and change from baseline of two viscoelastometric parameters: activated clotting time (ΔACT; ≥40s), and clot rate (CRpost; ≤20U/min). Following an initial injection at 1.3mg/kg, anti-Xa activity of 5/6 dogs reached or exceeded the target range. Following the final dose, anti-Xa activity reached or exceeded the target range in all dogs, and ΔACT and CRpost values exceeded target for 2-6 and 4-12h, respectively. At an enoxaparin dosage of 1.3mg/kg, SC, q8h, anti-Xa activity was consistently above the minimum threshold of the target range; however, the safety of this dosage remains to be determined.

Study of clinical and diagnostic parameters in patients with acute coronary syndrome who underwent COVID-19

The 2019 coronavirus infection pandemic (COVID-19) is widespread worldwide and is characterized by severe complications from the respiratory system, kidneys, liver, etc. The article presents an analysis of ACS in patients with COVID-19. It was revealed that in patients with coronavirus infection, acute coronary syndrome is characterized by a large number of complications and a high risk of death. The article presents the results of a study conducted by the authors on the study of acute coronary syndrome in patients who have undergone COVID-19. The study compares two groups of patients with ACS, depending on the presence of COVID-19 in the anamnesis. In the comparison groups, the analysis of the prevalence of risk factors for cardiovascular diseases was carried out: smoking, age, diabetes, obesity, dyslipidemia. The results of biochemical blood analysis and coagulogram were analyzed. It was found that patients with acute coronary syndrome who underwent COVID-19 were less likely to have risk factors for cardiovascular diseases. Patients with acute coronary syndrome who underwent COVID-19 had lower blood glucose, cholesterol, VLDL, triglycerides and higher blood clotting rates. As a result of the study, it was revealed that COVID-19 is an independent risk factor for the development of acute coronary syndrome against the background of multiple coronary vascular lesions.

Sonoclot Signature Analysis: A New Point-of-Care Testing Method for Defining Heat Stroke-Induced Coagulopathy.

PurposeData regarding the incidence of a coagulable state following heat stroke as assessed by Sonoclot signature analysis are limited. Our purpose was to appraise coagulopathy using a dynamic test capable of analyzing the entire coagulation cascade and to characterize coagulation in patients with heat stroke prior to transfusion.Materials and MethodsThe data of 106 patients were collected prospectively from the Critical Care Center of the General Hospital of Guangzhou Military Command. Coagulable state was defined as normal. Both hyper- and hypo-coagulable states were defined as coagulation defects. Hypercoagulability was defined as an activated clotting time (ACT) ≦195s and a clot rate (CR) >23, and hypocoagulability was defined as an ACT ≧119s and a CR < 7. The Sonoclot signature t examination was performed at the time of admission. Conventional tests, such as the prothrombin time (PT) and activated partial thromboplastin time (aPTT), were compared with Sonoclot monitoring to identify coagulation defects.ResultsThe average age of the 106 patients was 23.2±2.5 years. There were 102 males (96.3%) and 4 females (3.7%). Thirty-four patients (32.1%) were hypercoagulable and 44 patients (41.5%) were hypocoagulable at the time of admission; 28 patients (26.4%) had no evidence of a coagulopathy. Patients with hypocoagulability, unlike patients with hypercoagulability, had a higher sequential Organ Failure Assessment score, indicating a more severe multiple organ dysfunction score. Mortality was 5.9% in patients with hypercoagulability compared with 3.5% in patients with normal coagulation, and 18.1% in patients with a hypocoagulable state (P < 0.05). ACT was a predictor of mortality, while the CR and platelet function did not show statistical significance.ConclusionThis study determined the clinical outcomes and prognostic value of coagulability in patients with heat stroke, as defined by Sonoclot signature analysis at the time of admission.

Quantitative analysis of clot density, fibrin fiber radius, and protofibril packing in acute phase myocardial infarction

IntroductionCoronary artery disease is associated with impaired clot structure. The aim of this study was to investigate acute phase myocardial infarction (AMI) and provide detailed quantitative analysis of clot ultrastructure. Materials and methodsClot formation and breakdown, pore size, fiber density, fiber radius and protofibril packing were investigated in plasma clots from AMI patients. These data were compared to those from healthy controls. ResultsAnalysis on clot formation using turbidity showed increased lag time, suggesting changes in protofibril packing and increased fiber size for AMI patients compared to healthy controls. Additionally, increased average rate of clotting and decreased time to maximum absorbance in AMI patients suggest that clots formed more quickly. Moreover, we observed increased time from max OD to max rate of lysis. Increased fibrinogen and decreased plasminogen in AMI patients were accounted for in represented significant differences. AMI samples showed increased time to 25% and 50% lysis, but no change in 75% lysis, representative of delayed lysis onset, but expediated lysis once initiated. These data suggest that AMI patients formed less porous clots made from more densely packed fibers with decreased numbers of protofibrils, which was confirmed using decreased permeation and increased fiber density, and decreased turbidimetry. ConclusionsAMI plasma formed clots that were denser, less permeable, and lysed more slowly than healthy controls. These findings were confirmed by detailed analysis of clot ultrastructure, fiber size, and protofibril packing. Dense clot structures that are resistant to lysis may contribute to a prothrombotic milieu in AMI.

Bleeding Risk Assessment By Sonoclot in Severe Immune Thrombocytopenia

Patients of Immune Thrombocytopenia (ITP) are at increased risk of bleeding and the risk increases with decreasing platelet count. Although in patients of severe ITP (platelet < 30x109/L), the risk of bleeding is the highest yet not all patients bleed. If bleeders and non-bleeders in severe ITP can be reliably differentiated, the financial, physical and social burden can be significantly reduced. However, the available investigations have not been able to serve this purpose. Therefore, we included sonoclot; a global test of coagulation, as a tool to assess the risk of bleeding in ITP. Sonoclot coagulation and platelet function analyser uses viscoelastometry to measure coagulation. It provides information on the haemostasis process, including coagulation, fibrin gel formation, fibrinolysis, and is also able to assess platelet function. The sonoclot also has advantage of ability to measure platelet function in severe thrombocytopenia. It is a portable machine, which can be used as bedside tool in emergency out patient department (OPD) or for admitted patients.The study was conducted at department of internal medicine, postgraduate institute of medical education and research, Chandigarh, India. In this prospective observational study, a total of 80 patients of severe ITP (newly diagnosed) with or without active bleeding were included. Fifty of them had no evidence of any bleeding (Non-Bleeder Group) while thirty of them had features of (WHO grade ≥2) bleeding (Bleeder Group). All these patients were clinically evaluated and samples were collected as per unit protocol for ITP. They were evaluated with Hess test, bleeding time (BT), clotting time (CT), prothrombin time (PT), activated partial thromboplastin time (aPTT), and sonoclot signature analysis. For sonoclot; fresh blood samples were drawn in citrated vaccutainer and analysed by sonoclot coagulation and platelet function analyser made by Sienco, Inc, model SCP1, made in USA. The sonoclot signature assessment gives value for activated clotting time (ACT), clot rate (R1) and platelet function (PF).Both the groups were matched in terms of patient age and all had same diagnosis. The mean and standard deviation for variables were as in Table 1. The difference between platelet counts in two groups was non significant (p=.816). The difference between two groups when compared with Hess test (p=.21, INR (p=.794), bleeding time (p=.051) and clotting time (p=.058) was non-significant. However, significant difference in two sonoclot parameters was observed between two groups. ACT was significantly longer in the bleeders compared to non-bleeders (p=.009) and the platelet function measured by sonoclot signature analysis (depicted by R3) was significantly lower in the bleeders than the non-bleeders (p=.01) (Figure 1 and Figure 2). The clot rate (R1) was non-significantly lower in the bleeders.Although conventional evaluation tools like hess test, bleeding time, clotting time and INR could not differentiate between bleeders and non-bleeders in severe ITP, the coagulation and platelet function parameters assessed by Sonoclot coagulation and platelet function analyser were significantly different between bleeders and non-bleeders. Sonoclot also offers ease of use as compared to other global tests of coagulation (TEG/ ROTEM). Therefore, we conclude that to predict risk of bleeding in severe ITP patients; to help clinician in being more conservative in management of severe ITP patients with low risk of bleeding and avoid unnecessary therapy, sonoclot signature analysis is a handy outpatient or bedside tool. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Utility of the Sonoclot analyzer to assess hyperfibrinolysis in dogs.

Coagulation abnormalities, including hyperfibrinolysis, have been documented in sick veterinary patients. Viscoelastic tests, including the Sonoclot Coagulation and Platelet Function Analyzer, are useful in detecting hyperfibrinolysis. Tissue plasminogen activator (tPA) assays have been used to quantify fibrinolysis using thromboelastography. We aimed to document and evaluate changes in the whole blood of healthy dogs exposed to in vitro tPA at varying concentrations using the Sonoclot analyzer. Ten milliliters of blood was collected from healthy adult dogs. Sonoclot tests were run in duplicate and included a control sample and five tPA concentrations: 50, 75, 100, 150, and 200IU/mL of blood. Eleven dogs were enrolled in the study. Based on standard Sonoclot Signature changes, a numeric value fibrinolysis time (FTi) was derived to aid in the quantification of hyperfibrinolysis. Activated clotting time and clot rate Sonoclot values were not significantly affected by any tPA concentration. There was a significant decrease in platelet function (PF) at tPA concentrations equal to and above 75IU/mL on channel 1 and tPA concentrations of 150IU/mL and higher on channel 2. There was a progressive decrease in FTi at increasing tPA concentrations. The Sonoclot analyzer can be used to evaluate hyperfibrinolysis. Predictable changes were seen in the Sonoclot Signature and a decrease in PF and FTi was found with increasing tPA concentrations. The Sonoclot assay with a tPA concentration of 100IU/mL is suggested a baseline measure of hyperfibrinolysis and has a resultant median FTi of 42minutes, which is a practical time for clinical applications.

Diclofenac Is Superior to Paracetamol in Postoperative Pain Scores and Analgesic Consumption in Supratentorial Craniotomy With No Difference in Platelet and Clot Function: A Prospective Randomized Controlled Trial.

The use of nonsteroidal anti-inflammatory drugs in neurosurgery remains controversial because of potential risk of hematoma formation secondary to platelet dysfunction. This study aimed to investigate the efficacy and safety of diclofenac compared with paracetamol for the management of postcraniotomy pain. In all, 110 adult patients undergoing craniotomy for supratentorial tumors were randomized to receive either intravenous paracetamol (15 mg/kg) or intravenous diclofenac sodium (1.5 mg/kg) 30 minutes before the end of surgery and postoperatively at 12-hour intervals up to 48 hours. The analgesic efficacy of diclofenac and paracetamol was assessed using the Numerical Rating Scale (NRS) score at 12, 24, and 48 hours after surgery. We also examined the need for rescue analgesia, coagulation profiles using a Sonoclot analyzer, and incidence of intracranial hematoma formation. At 24 hours postsurgery, NRS scores were lower in group D than in group P; median (interquartile range) and mean rank NRS score in group D was 3.00 (1.0), 43.71, respectively, compared with 3.00 (1.0), 59.29 in group P (P=0.004). Patients in group P received more rescue analgesia than those in group D. Coagulation profiles were similar between groups at 24 hours. Activated clotting time was longer in group D (128.76±12.61 s) than in group P (123.84±09.77 s; P=0.03) at 48 hours, although remained within normal limits in both groups; clot rate and platelet function were similar at 48 hours. There was no difference in the incidence of postoperative tumor bed hematoma. Compared with paracetamol, diclofenac sodium provided more effective postoperative analgesia at 24 hours with no evidence of adverse effects on coagulation profiles in patients undergoing craniotomy for supratentorial tumors.

Clinical Validation of Global Coagulation Tests to Guide Blood Component Transfusions in Cirrhosis and ACLF.

Background and AimsPatients with cirrhosis and acute-on-chronic liver failure (ACLF) may have bleeding complications and need for invasive procedures. Point-of-care (POC) coagulation tests like thromboelastography (TEG) and Sonoclot may be better for guiding patient management than the standard coagulation tests (SCTs), like prothrombin time, platelet count and international normalized ratio.MethodsWe prospectively compared and validated the POC tests and SCTs in 70 persons with ACLF and 72 persons with decompensated cirrhosis who had clinical bleeding and checked for episodes of re-bleeding and transfusion requirements. We assessed pre-procedure requirement of blood components when correction was done based on an SCT or POC strategy.ResultsEpisodes of bleeding were seen in 45% and 28% of ACLF and cirrhosis patient, respectively (p=0.036), with the major site of bleeding being gastrointestinal (31% and 16%, respectively). Platelet counts correlated with TEG-maximum amplitude in cirrhosis (p=0.045) and prothrombin time correlated positively with TEG-reaction (R) time (p=0.032), TEG-Clot kinetics (K) time (p=0.042), Son-activated clotting time (p=0.038) and negatively with clot rate (p=0.043) in ACLF, making these correctable target variables in POC transfusion algorithms. Of 223 procedures, transfusion of fresh frozen plasma and platelet concentrate was reduced by 25% (p=0.035) and 20.8% (p=0.045) by using a POC strategy in 76 patients. Correction of deranged Son-activated clotting time and TEG-reaction time was noted in 68% and 72% after 24 h of fresh frozen plasma transfusion in ACLF and 85% and 80% in cirrhosis, respectively.ConclusionsOur study clinically validates that POC tests can better detect coagulation defects and transfusion thresholds in ACLF and cirrhosis, whereas use of conventional tests appear to be less suitable in patients with clinical bleeding.Trial RegistrationNCT04332484.

ОЦІНКА ВПЛИВУ ДОНОРА МОНООКСИДУ КАРБОНУ (CORM-2) НА ШВИДКІСТЬ ЗГОРТАННЯ КРОВІ В УМОВАХ ІМУННОЇ ВІДПОВІДІ

The balance between the liquid state support of the blood and the thrombus formation process depends on the activity of plasma factors and thrombocytes. In a vascular injury condition, thrombocytes form a thrombocyte plug, vasoconstriction and, further, take part in the restoration of the tissues. Reducing the clotting function of thrombocytes is a current problem. The search for agents - modulators of thrombocyte activity - is a promising area of research. Compounds - gas-transmitters can be used to model the activity of thrombocytes hemostasis. Carbon monoxide (CO) gas belongs to this group as well. CO in very low concentrations affects in a different way the process of apoptosis, stimulates calcium-dependent potassium channels, and changes the activity of mitochondria. To investigate the effect of CO on blood clotting rate, laboratory mice were injected with the compound CO donor (CORM-2). This substance was administered separately during the inductive and productive phases of the immune response. The dynamics of the immune response were measured by IgA, IgM and IgG immunoglobulin levels. It was found that blood clotting was intensified under the influence of CORM-2 (20 mg/kg). Especially, the increase was during the productive phase. At the end of the experiment, the percentage of megakaryoblasts in the total population of thrombocyte precursors decreased in the bone marrow of animals (to which CORM-2 was administered). Against this background, an increased content of megakaryocytes was found in the group that received CORM-2 during the productive phase. In the group that received CORM-2 during the inductive phase, in addition to an increase in megakaryocyte levels, there was an increase in thrombocyte levels. The group that received CORM-2 during the productive phase also had a predominance of metamegakaryocytes and reducing of thrombocyte amount. Key words: thrombocytes, carbon monoxide donor, CORM-2, immune response phases.

Hypocoagulable Coagulation Profile and Endogenous Heparinoids Are Associated with Invasive Ventilation and Mortality in COVID-19

Background & Aims: A procoagulant state with pulmonary micro-thrombosis has been described in COVID-19, which contributes to pathophysiology of respiratory failure along with diffuse alveolar damage. Cytokine storm and systemic inflammation in COVID-19 generates endogenous heparinoids from the endothelium which leads to secondary organ failures. We prospectively studied the dynamic association of the coagulation abnormalities with respiratory failure and mortality in patients with COVID-19.Methods: We did a prospective observational study in adults aged between 18-80 years with COVID 19 pneumonia requiring intensive care (ICU) admissions in a tertiary care hospital in North India. All patients received standard medical therapy based on best available evidence. Patients were categorized based on the oxygen support therapy into low flow, high flow and invasive ventilation groups. In patients considered to be at risk of thromboembolic or bleeding phenomena, paired coagulation tests were performed at days 1 and 3 with, Sonoclot ® (glass beaded and heparinase-treated). All patients were consecutively followed for inflammatory markers, ventilator requirements and survival. Activated clotting time (ACT) 75 units were used as the cut-off for hypercoagulable state. The heparin like effect (HLE) was considered by a correction of ≥ 40 s in hACT (heparinized treated ACT). The trial is registered with ClinicalTrials.gov (NCT04668404).Results: A total of 2324 patients with COVID-19 were screened from August 2020 to November 2020. Two hundred and fifteen patients requiring ICU admission were assessed and 74 patients with a median age of 54(42-67) years were enrolled. A procoagulant profile was seen in 45.5%, 32.4% and 20.7% in low-flow, high-flow, and invasive ventilation. Paired Sonoclot ® assays in 33 patients demonstrated HLE in 17 (51.5%) and 20 (62.5%) at days 1 and 3 respectively. In multivariate analysis, the presence of HLE (HR 1.02; CI 1.08-1.6; p 131 s, clot rate 96.7 mg/l at admission predicted mortality.Conclusions: HLE contributes to hypocoagulable effect, need for invasive ventilation and mortality in patients with COVID-19.Trial Registration: The trial is registered with ClinicalTrials.gov (NCT04668404).Funding Statement: NoneDeclaration of Interests: NoneEthics Approval Statement: Ethical clearance was obtained from the Institutional Review Board (PGI/IEC/2020/000997 dated 24 August 2020)

Intense light as anticoagulant therapy in humans.

Blood coagulation is central to myocardial ischemia and reperfusion (IR) injury. Studies on the light elicited circadian rhythm protein Period 2 (PER2) using whole body Per2-/- mice found deficient platelet function and reduced clotting which would be expected to protect from myocardial IR-injury. In contrast, intense light induction of PER2 protected from myocardial IR-injury while Per2 deficiency was detrimental. Based on these conflicting data, we sought to evaluate the role of platelet specific PER2 in coagulation and myocardial ischemia and reperfusion injury. We demonstrated that platelets from mice with tissue-specific deletion of Per2 in the megakaryocyte lineage (Per2loxP/loxP-PF4-CRE) significantly clot faster than platelets from control mice. We further found increases in infarct sizes or plasma troponin levels in Per2loxP/loxP-PF4-CRE mice when compared to controls. As intense light increases PER2 protein in human tissues, we also performed translational studies and tested the effects of intense light therapy on coagulation in healthy human subjects. Our human studies revealed that intense light therapy repressed procoagulant pathways in human plasma samples and significantly reduced the clot rate. Based on these results we conclude that intense light elicited PER2 has an inhibitory function on platelet aggregation in mice. Further, we suggest intense light as a novel therapy to prevent or treat clotting in a clinical setting.

Clot formation, lysis and ultrastructure in patients with acute phase myocardial infarction

Abstract Background/Introduction Patients with established coronary artery disease have impaired clot structure and lysis. Prior research has compared patients with myocardial infarction to those with stable coronary artery disease (without a healthy reference), or patients after myocardial ischemia. The role of clot structure in the acute phase of the disease is unknown. Purpose Investigate clot structure in the acute phase of myocardial infarction (AMI) compared to healthy controls. Methods Plasma clots from AMI patients (within 12 hours of symptoms onset) or healthy controls were analyzed by turbidity and lysis. Clot pore size was determined by permeation. Clot ultrastructure was determined by confocal and scanning electron microscopy. Average fiber radius and protofibril packing were investigated by turbidimetry. Results Analysis on clot formation, using turbidity, showed increased lag time (24%, p&amp;lt;0.05), suggesting changes in protofibril packing and increased fiber size for AMI patients compared to controls. Additionally, AMI clots formed more quickly as evidenced by average rate of clotting (2.9-fold increase, p&amp;lt;0.001) and time to maximum absorbance (38% decrease, p&amp;lt;0.0001). Decreased plasminogen in AMI patients (23%, p&amp;lt;0.05) had strong effects on clot lysis, including increased time to half lysis (1.72-fold, p&amp;lt;0.0001) and increased time to maximum lysis rate (7.96-fold, p&amp;lt;0.0001). Taken together, these results suggest that AMI patients form less porous clots made from densely packed fibers with decreased numbers of protofibrils, which was confirmed using permeation (1.05-fold decrease, p&amp;lt;0.0001), confocal (Figure 1) and scanning electron microscopy (46% increase, p&amp;lt;0.0001), and turbidimetry (16% decrease, p&amp;lt;0.05) respectively. Conclusions Plasma from patients with AMI form denser and less permeable clots that lyse more slowly than healthy controls. These data show marked abnormalities in clot ultrastructure that contribute to the high risk of thrombosis during the acute phase of myocardial infarction, which has not shown in previous investigations. Figure 1. Confocal microscopy of AMI vs. controls Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Jagiellonian University Medical College to G.G. (N41/DBS/000096) and BHF Programme Grant to S.R.B. and R.A.S.A. (RG/18/11/34036).

The effect of hematocrit, fibrinogen concentration and temperature on the kinetics of clot formation of whole blood.

Dynamic mechanical analysis of blood clots can be used to detect the coagulability of blood. We investigated the kinetics of clot formation by changing several blood components, and we looked into the clot "signature" at its equilibrium state by using viscoelastic and dielectric protocols. Oscillating shear rheometry, ROTEM, and a dielectro-rheological device was used. In fibrinogen- spiked samples we found the classical high clotting ability: shortened onset, faster rate of clotting, and higher plateau stiffness. Electron microscopy explained the gain of stiffness. Incorporated RBCs weakened the clots. Reduction of temperature during the clotting process supported the development of high moduli by providing more time for fiber assembly. But at low HCT, clot firmness could be increased by elevating the temperature from 32 to 37°C. In contrast, when the fibrinogen concentration was modified, acceleration of clotting via temperature always reduced clot stiffness, whatever the initial fibrinogen concentration. Electrical resistance increased continuously during clotting; loss tangent (D) (relaxation frequency 249 kHz) decreased when clots became denser: fewer dipoles contributed to the relaxation process. The relaxation peak (Dmax) shifted to lower frequencies at higher platelet count. Increasing temperature accelerates clot formation but weakens clots. Rheometry and ROTEM correlate well.

Investigating the impact of M1 protein from Group A Streptococcus on fibrin clot formation, structure and fibrinolytic potential

Background: Fibrin formation is an essential part of innate immunity, sealing off infections to limit bacterial spreading.The surface-anchored M1 protein is a major virulence determinant of Group A streptococcus (GAS). During early infection M1 is cleaved from the cell surface by SpeB, a streptococcal protease regulated by CovR/S. M1 forms a supramolecular complex with fibrinogen however the impact on fibrin formation is not known. Methods: The effects of recombinant M1 (rM1) were assessed in fibrin clots made from plasma or purified fibrinogen incubated with thrombin, by confocal microscopy and scanning electron microscopy. Clotting and lysis profiles (with plasminogen activators and plasminogen) were investigated kinetically using thromboelastography (ROTEM). Results: rM1 (0.47-60 μg/ml) increased clotting rates to produce heterogeneous clots with irregular fibre bundles and compacted fibrin. Formation of the protective fibrin biofilm was also disrupted by rM1. Furthermore, mechanical strength of fibrin clots was reduced with increasing rM1 concentrations and was undetectable above 15.5 μg/ml. Purified and plasma clots formed with rM1 were more susceptible to lysis by plasmin, with a 1.4 – 2-fold reduction in lysis times. Conclusions: At sites of GAS infection, cleaved M1 may bind to fibrinogen generating fibrin clots which: lack the protective film at the clot surface; are mechanically weaker; and are less resistant to lysis by plasmin. GAS strains of M1-type are commonly associated with invasive infections; the impact of M1 on fibrin structure could contribute to the severity of GAS infection by compromising the fibrin barrier that limits bacterial proliferation and migration.

Viscoelastic test-based bleeding risk score reliably predicts coagulopathic bleeding in decompensated cirrhosis and ACLF patients.

Coagulopathic bleeding risk prediction is challenging in decompensated cirrhosis (DC) by conventional assays. Viscoelastic tests (VETs) are likely to be more useful for assessing coagulation status in these patients. We investigated whether the VET (Sonoclot) parameters with fibrinogen could predict coagulopathic bleeding in these patients. Coagulation parameters studied in 874 patients [124 compensated cirrhosis (CC), 521 DC, and 229 acute-on-chronic liver failure (ACLF)] and 190 controls. DC patients were enrolled in derivation (n = 266) and validation (n = 255) cohorts. Sonoclot variables [activated clotting time (ACT), clot rate (CR), platelet function (PF), time to peak (TP) and peak amplitude (PA)] and fibrinogen levels were measured. Coagulopathic bleeding was recorded along with 1-year survival. DC patients had prolonged ACT (p < 0.001), depressed CR (p = 0.059), reduced PF (p = 0.09), longer TP (p < 0.001) and smaller PA (p < 0.001), compared to CC and controls (p < 0.001 each). In derivation cohort, 32.3% patients had coagulopathic bleeding. Cox regression analysis of derivation cohort revealed; ACT > 190s, PF < 1.25 and fibrinogen < 1.2g/l could predict coagulopathic bleeding and were used to develop a bleeding risk score. In validation cohort; this score was comparable, correlated to real events, and had similar bleed free events with time. The score was also useful in predicting bleed in ACLF patients. In DC patients, 1-year mortality was higher those who bled and received transfusions. Viscoelasticity-based bleeding risk score using ACT, PF and fibrinogen, predicts coagulopathic bleeding in DC patients and should be useful in rationalizing transfusion of blood products. We designed a viscoelastic test-based bleeding risk score which is useful in advanced liver disease to predict the coagulation-related bleeding. This figure shows the lower bleeding-free events in advanced cirrhosis with each incremental bleeding risk score.

Continuous Renal Replacement Therapy in Venovenous Extracorporeal Membrane Oxygenation: A Retrospective Study on Regional Citrate Anticoagulation.

Systemic infusion of unfractionated heparin (UFH) is the standard anticoagulation technique for continuous renal replacement therapy (CRRT) during extracorporeal membrane oxygenation (ECMO), but often fails to avoid CRRT circuit clotting. The aim of this study was to assess, in patients undergoing CRRT during venovenous ECMO (vv-ECMO), the efficacy and safety of adding regional citrate anticoagulation (RCA) for CRRT circuit anticoagulation (RCA + UFH group) compared with the sole systemic heparin anticoagulation (UFH group). We performed a retrospective chart review (2009-2018) of patients treated with CRRT during ECMO. We evaluated filter life span, rate of CRRT circuit clotting, and coagulation parameters. The incidence of citrate anticoagulation-related complications was recorded. Forty-eight consecutive adult patients underwent CRRT during vv-ECMO in the study period. The incidence of CRRT circuit clotting was lower in the RCA + UFH group (11% vs. 38% in the UFH group, p < 0.001). Log-rank survival analysis demonstrated longer circuit lifetime for RCA + UFH group. No complication ascribable to citrate anticoagulation was recorded. Regional citrate anticoagulation resulted a feasible, safe, and effective technique as additional anticoagulation for CRRT circuits during ECMO. Compared with systemic heparinization only, this technique allowed to reduce the rate of CRRT circuit clotting.

Chitosan/Ulva lactuca polysaccharide hydrogel containing copper (ii) beneficial for biofilm-associated wound infection: Formulation characterizations and In vitro study

The present investigation elaborates the energy efficient synthesis of chitosan/Ulva lactuca polysaccharide hydrogel impregnated with copper (II) (Cu-CS/ULP) for wound dressing applications. We successfully characterized the prepared hydrogel using FTIR, SEM, TG-DTA and swelling analysis. Compare to chitosan/U.lactuca polysaccharide hydrogel (CS/ULP), the Cu-CS/ULP hydrogel show a similar framework with the best mechanical properties. The whole blood clotting examination shows that the copper (II) can expand the blood clotting rates of the CS/ULP hydrogel. Free radical scavenging activity and prevention of protein degradation confirmed by DPPH and protein degradation assays. The released copper (II) from Cu-CS/ULP hydrogel exhibited a strong antibacterial activity against Gram-negative (Escherichia coli and Klebsiella pneumoniae) and Gram-positive (Bacillus subtilis and Staphylococcus aureus). Cu-CS/ULP hydrogel showed a significant activity in biofilm inhibition mass compared to that of CS/ULP. The present suggested that the copper (II) loaded CS/ULP hydrogel is a promising biomaterial for wound dressing application.