Platelet Fibrinogen Research Articles

POST-OPERATIVE COMPLICATIONS IN CARDIAC SURGERY PATIENTS

TOPIC: Cardiothoracic Surgery TYPE: Original Investigations PURPOSE: Perioperative coagulopathy and postoperative bleeding are the most common complications in patients undergoing cardiac surgery, especially when the cardiovascular surgery is associated with cardiopulmonary bypass (CPB). In this context, some studies suggest that implementation of viscoelastic point-of-care tests (POCT), such as rotational thrombo-elastometry, in conjunction with a specific algorithm for coagulation management in cardiac surgery, allow for better control of hemostatic pathology. METHODS: Retrospective cohort study including 675 patients who underwent cardiac surgery with cardiopulmonary bypass. This first group (N=336) is characterized by having received perioperative transfusion therapy based on conventional laboratory coagulation tests and clinical judgment. This second group (n=339) is characterized by having received perioperative transfusion therapy based on the results of the ROTEM® viscoelastic point-of-care test system and a specific coagulation management algorithm. RESULTS: Following viscoelastic testing and the implementation, the incidence of any allogeneic blood transfusion decreased (41.4% vs 31.9%, p=0.026) during the perioperative period. In the group monitored with ROTEM®, decreased incidence of transfusion was observed for packed red blood cells (31.3% vs 19.8%, p=0.002) and fresh frozen plasma (9.8% vs 3.8%, p=0.008). Increased incidence was observed for platelet transfusion (4.8% vs 6.8%, p=0,530) and fibrinogen concentrate (0.9% vs 3.5%, p=0.066). Similar results were observed in the postoperative period, but with a decreased incidence of platelet transfusion (4.8% vs 3.8%, p=0.813). In addition, statistically significant reductions were detected in the incidence of postoperative bleeding (9.5% vs 5.3%, p=0.037) and surgical reexploration (6.0% vs 2.9%, p=0.035).Thus, the incidence of cardiac surgery complications in ICU decreased (55.8% vs 57.7%, p=0.275), especially acute postoperative pericarditis (3.6% vs 1.2%, p=0.043).Finally, we observed a lower rate of ICU readmissions for viscoelastic testing patients (4.6% VS 2.7%, p=0.201); and statistically significant reductions were detected in length of Intensive Care Unit (ICU) stay (6.0 days vs 5.3 days, p=0.026).In addition, we observed a decreased in total hospital stay (16.7 ±10.0 days vs 18.3±13.5 days, p=0.076), and in hospital mortality associated with cardiac surgery (4.5% vs 2.4%, p=0.132). CONCLUSIONS: The monitoring of hemostasis by ROTEM® in cardiac surgery, was associated with decreased incidence of allogeneic blood transfusion, clinical hematologic and cardiac postoperative complications and lengths of ICU stay. CLINICAL IMPLICATIONS: Finally, we have achieved great results, including decreased incidence of blood transfusion, decreased complications, lower lengths of stay in the ICU and a reduction cardiac surgery hospital cost (622,008 euros vs 312,750 euros). after ROTEM implementation. DISCLOSURES: No relevant relationships by Isabel Rodríguez Martín, source=Web Response

Effect of Diabetes Mellitus and Hypertension on Osmotic Fragility and Hemorheological Factors in Male Wistar Rats

Diabetes mellitus is a common risk factor for erythrocyte osmotic stress. This study was aimed at exploring the effect of streptozotocin (STZ)-induced diabetes mellitus and salt-induced hypertension on osmotic fragility and hemorheological variables in male Wistar rats. Thirty male rats were grouped into five groups of six animals each as follows: negative control (zero salt in diet); positive control (normal salt diet - 0.3% salt); high salt diet (8% salt) (HSD only); STZ induced diabetes and normal salt diet (STZ only); STZ induced diabetes and high salt diet (STZ + HSD). At the end of a 4 weeks period, hematological variables, osmotic fragility, rheology and cardiovascular responses were assessed. There was an increase (p<0.05) in the mean arterial pressure and heart rate of HSD, STZ and HSD + STZ groups indicating a salt induced hypertension. There was a decrease in the body weight of STZ and HSD +STZ groups. There was significant increase (p<0.05) in the haematocrit, platelets estimates and fibrinogen concentrations in the experimental groups when compared with the controls. The STZ and STZ + HSD groups showed a reduced clotting time which corresponded to the increased platelet estimates and fibrinogen concentration. The increase in haematocrit, platelet and plasma protein resulted in the increased blood viscosity and a decreased flow rate. The osmotic fragility test was also observed to be increased (p<0.05) in HSD, STZ only and STZ + HSD groups. Diabetes mellitus and hypertension increase the rate of hemolysis of erythrocyte, as well as increase blood viscosity.

Clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia

The aimof the study was to assess the clinical and genetic factors associated with the risk of recurrent ischemic events in patients with stable stenocardia (SS).Materials and methods.A total of 100 patients with SS were examined and followed-up for 15.3±8.3 months. The patients were divided into subgroups (SG): SG1 (n=51) – persons without events, SG2 (n=49) persons with recurrent ischemic events (hospitalization due to the development of pain syndrome, re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes), SGB (n=11) – persons with «major» recurrent ischemic events (re-stenting due to stent restenosis, myocardial infarction, cerebral infarction and death from cardiovascular causes) , SGG (n=89) – persons without «major» events. The obtained survey data (general clinical, aggregometry, polymor phism of genes of platelet fibrinogen receptor ITGB3 (T1565C), platelet collagen receptor ITGA2 (C807T), ADP platelet receptor P2RY12, H1/H2 (T744C)) were analyzed using the STATISTICA 10.0 software.Results.In SG2, men predominated (χ2=9.2; p<0.01), past MI was more common (χ2=4.8; p<0.05), more stents were implanted (2.4±1.9 versus 1.7±1.1, p<0.05), TRAP-test values were higher (p<0.05) compared to SG1. In SGB, greater number of stents were implanted (3.1±2.2 versus 1.61±1.57, p<0.05), the carriage of the TC genotype of the ITGB3 gene was more common, (p<0.05), a combination of gene mutations ITGB3 and P2RY12 was more common, (p<0.05) compared to SGG. A logistic regression equation was constructed, including the presence of diabetes mellitus, the number of platelets in the blood test, the ASPI-test values, the carriage of the 1565C allele of the ITGB3 gene, the number of stents implanted, which makes it possible to determine the likelihood of developing «major» recurrent ischemic events with a cut-off threshold LP₀=0.0965, with sensitivity – 81.82 %, specificity – 78.48 %, overall accuracy – 78.89 %.Conclusions.The factors associated with the development of recurrent ischemic events are: male sex, previous MI, a greater number of implanted stents, and high TRAP-test values. The factors associated with the development of recurrent «major» ischemic events are: a greater number of implanted stents, carriage of the TC genotype of the ITGB3 gene, carriage of a combination of mutations of the H1/H2 polymorphic locus of the P2RY12 gene and the T1565C polymorphic locus of the ITGB3 gene, diabetes mellitus, the number of platelets in blood test, ASPI-test values.

Rumex acetosella Inhibits Platelet Function via Impaired MAPK and Phosphoinositide 3-Kinase Signaling.

To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract. Standard light aggregometry was used for platelet aggregation, intracellular calcium mobilization assessed using Fura-2/AM, granule secretion (ATP release) by luminometer, and fibrinogen binding to integrin αIIbβ3 detected using flow cytometry. Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling. Rumex acetosella displayed the ability to inhibit platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin αIIbβ3. Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation (all P<0.01). Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling, and it may protect against the development of platelet-related cardiovascular diseases.

166 CLINICAL IMPACT OF NEW BIOMARKERS IN THE PATIENTS WITH ESOPHAGEAL CARCINOMA

Abstract We have developed several new biomarkers in patients with esophageal carcinoma. In this paper, we reviewed the clinical impact of serum autoantibodies, growth factors, blood counts, and other serum tumor markers. Methods Blood samples of patients with esophageal carcinoma were obtained before surgery and were stored at −80°C until the ELISA assay. Target tumor antigens for autoantibodies were SURF1, LOC, HOOK2, AGENCOURT, TROP-2, TRIM21, GLUT-1, Myomegalin, Makorin-1, CUEC-23, ECSA-1, p53, and NY-ESO-1. Growth factor-related molecules were followed; midkine, VEGF, PDECGF, and HGF. The other markers were followed; platelet, CRP, IAP, SCC-Ag, CYFRA21-1, and fibrinogen. Results A total of 24 biomarkers were evaluated for their clinical impact on patients’ survival and tumor stages. High positive rates, more than 40% were found in SURF1, Myomegalin, midkine, and HGF. Although the majority of autoantibodies did not have a prognostic impact, all the growth factors have a prognostic impact on patients’ survival. Lutine blood tests, such as platelet count, neutrophil-lymphocyte ratio, fibrinogen, and CRP, were also useful to predict prognosis. Conclusion We have evaluated the clinical impact of 24 new biomarkers, including 13 autoantibodies, for the patients with esophageal carcinoma. Although the autoantibodies were useful to detect relatively early stage of the disease, the prognostic impact of these biomarkers was limited. All growth factors were useful to predict patients’ survival.

Effect of Hemodilution &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; on the Hemostatic System

The aim of the study was to determine experimentally the effect of hemodilution by the 2:1 sterofundin/gelofusine (SG) solution on hemostatic parameters in vitro and in vivo.Material and methods. Experiments were carried out on 75 male Wistar rats weighing 270–380 g and anesthetized with intramuscular tiletamine-zolazepam (40 mg/kg) + xylazine (10 mg/kg). Animals were divided randomly into 4 groups: Group 1 — in vitro 25-percent dilution of carotid blood samples by the SG solution (n=12), Group 2 — in vitro 37.5-percent dilution of similar samples (n=11), Group 3 — in vivo 25-percent dilution (n=10), Group 4 — the controls (n=42) with no dilution. The first stage of the study compared the in vitro dilution groups with the control group and with each other; the second stage compared the in vivo dilution group with the control group. The parameters of low-frequency piezoelectric tromboelastography (LFPTEG), clotting tests and complete blood count were studied to evaluate the effect of hemodilution.Results. At a 25-percent hemodilution with 2:1 CG solution in vitro and in vivo, the hemostatic parameters retained within the reference limits, but a trend to increased intensity of the enzymatic reactions of the coagulation cascade and a significant increase in clot polymerization in vitro due to relative anticoagulant deficiency became evident. In vitro 37.5-percent blood dilution significantly reduced the blood level of fibrinogen and platelet count, inhibited the intensity of the proteolytic stage of coagulation, reduced the clot density at the T3 gelation point, at 5 minutes after reaching it and the maximum amplitude (MA) of the LFPTEG curve, as well as significantly reduced anticoagulant activity of the blood. The observed changes in hemostatic parameters were significantly outside the reference limits, which may affect the interpretation of the experimental results and be clinically important. We found negative correlation between clot density and platelet activity at 25-percent dilution in vivo, whereas at 37.5-percent dilution in vitro an additional positive correlations between platelet count and fibrinogen levels were determined.Conclusion. A 25-percent hemodilution with 2:1 CG solution should be considered «safe» for the in vivo hemostatic system providing minimal effect on the in vitro parameters in the exper-iment.

Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential.

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

Adenovirus-Vectored COVID-19 Vaccine-Induced Immune Thrombosis of Carotid Artery: A Case Report.

Venous thrombosis and thrombocytopenia after vaccination with the adenovirus-vectored COVID-19 vaccine ChAdOx1 nCov-19 (AstraZeneca) have been linked to serum antibodies against platelet factor 4 (PF4)-polyanion complexes. We here report vaccine-induced isolated carotid arterial thrombosis. Imaging and laboratory findings, treatment decisions, and outcome of this case are presented. Eight days after having received the first dose of ChAdOx1 nCov-19 vaccine, a 31-year-old man was admitted to our stroke unit with acute headache, aphasia, and hemiparesis. D-dimers were slightly elevated, but platelet count and fibrinogen level were normal. MRI-confirmed mainstem occlusion of middle cerebral artery resolved within 1 hour after the start of IV thrombolysis. A wall-adherent, nonoccluding thrombus in the ipsilateral carotid bulb was identified as the source of embolism. Cardiac or paradoxical (venous) embolism was excluded. Screening for the presence of heparin-induced thrombocytopenia-related antibodies was positive, and highly elevated serum IgG antibodies against PF4-polyanion complexes were subsequently proven. Treatment with aspirin and subcutaneous danaparoid, followed by phenprocoumon, led to thrombus shrinkage and dissolution within 19 days and favorable clinical outcome. Vaccine history is important in patients not only with venous but also with arterial thromboembolic events. Vaccine-induced immune thrombosis of brain-supplying arteries may well be handled.

Large hemorrhagic stroke after ChAdOx1 nCoV‐19 vaccination: A case report

BackgroundThe anti‐COVID‐19 vaccines are new, and we should be alert of potential adverse effects of them.Aims of the StudyTo report a case of a large hemorrhagic stroke 5 days after ChAdOx1 nCoV‐19 vaccination.MethodsClinical, imaging, and laboratory data of a patient with stroke admitted to our emergency department.ResultsA woman, 57 years old, took the first dose of the ChAdOx1 nCoV‐19 vaccine and shortly thereafter presented mild systemic symptoms and started on aspirin. On day 5, she had a sudden onset of sweating and paleness, which has followed by left hemiparesis, vomiting, and somnolence. Computed tomography showed a large right deep frontal lobe parenchymal hematoma with the inundation of the entire ventricular system. Platelets count, fibrinogen, prothrombin time, and D‐dimer were normal. Digital subtraction angiography did not show any signs of thrombosis or aneurysms in brain circulation.ConclusionsClinicians should be aware of cerebrovascular adverse effects of ChAdOx1 nCoV‐19, including out‐of‐context of vaccine‐induced immune thrombotic thrombocytopenia.

Extracorporeal Membrane Oxygenation Induces Early Alterations in Coagulation and Fibrinolysis Profiles in COVID-19 Patients with Acute Respiratory Distress Syndrome.

Hemostatic changes induced by extracorporeal membrane oxygenation (ECMO) support have been yet poorly documented in coronavirus-19 (COVID-19) patients who have a baseline complex hypercoagulable state. In this prospective monocentric study of patients with severe acute respiratory distress syndrome (ARDS) rescued by ECMO, we performed longitudinal measurements of coagulation and fibrinolysis markers throughout the course of ECMO support in 20 COVID-19 and 10 non-COVID-19 patients. Blood was sampled before and then 24 hours, 7, and 14 days after ECMO implantation. Clinical outcomes were prospectively assessed until discharge from the intensive care unit or death. The median age of participants was 47 (35-56) years, with a median body mass index of 30 (27-35) kg/m2, and a Sepsis-related Organ Failure Assessment score of 12 (8-16). Baseline levels of von Willebrand factor, fibrinogen, factor VIII, prothrombin F1 + 2, thrombin-antithrombin, D-dimer, and plasminogen activator inhibitor-1 (PAI-1) were elevated in both COVID-19 and non-COVID-19 ARDS patients, indicating that endothelial activation, endogenous thrombin generation, and fibrinolysis shutdown occur in all ARDS patients before ECMO implantation. From baseline to day 7, thrombin generation (prothrombin F1 + 2, p < 0.01) and fibrin formation markers (fibrin monomers, p < 0.001) significantly increased, further resulting in significant decreases in platelet count (p < 0.0001) and fibrinogen level (p < 0.001). PAI-1 levels significantly decreased from baseline to day 7 (p < 0.0001) in all ARDS patients. These changes were more marked in COVID-19 patients, resulting in 14 nonfatal and 3 fatal bleeding. Additional studies are warranted to determine whether monitoring of thrombin generation and fibrinolysis markers might help to early predict bleeding complications in COVID-19 patients supported by ECMO.

Prediction of intrahepatic cholestasis of pregnancy in the first 20 weeks of pregnancy

Aim Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disease associated with a significant risk of fetal complications including pre-term delivery and fetal death. Typically, it was diagnosed in the third trimester of pregnancy. This study utilized characteristics from routine maternal examinations in the first 20 weeks’ gestation to predict ICP in pregnant women. Methods This is a retrospective case-control study. 13,329 medical records were collected on pregnant women presenting to the West China Second University Hospital between December 2017 and December 2018. After screening according to strict criteria, a total of 487 patients, 250 intrahepatic cholestasis of pregnancy cases, and 237 controls were selected for this study. We collected seven maternal characteristics indices for analysis and forty-three routine blood examination indices were obtained from routine hepatic, renal, and coagulation function examinations. The least absolute shrinkage and selection operator regression was applied for variable selection. Classification and regression trees, logistic regression, random forests, and light gradient boosting machines were fit for predictive modeling. We randomly divided 25% of the original data as testing set to conduct internal validation of the performance of the prediction model. The area under the receiver operating characteristic curves (AUC) was used to compare methods. Results Eight variables were selected out as potentially significant predictors that could reliably predict ICP. The sensitivity, specificity, accuracy, and AUC of the final prediction model obtained by light gradient boosting machines were 72.41, 79.69, 76.23, and 79.77%, respectively. Significantly higher platelet large cell ratio, alanine aminotransferase, glutamyl transpeptidase, and fibrinogen levels were found in cases as compared to healthy controls, while activated partial thromboplastin time and mean corpuscular hemoglobin concentration levels were significantly lower (p < .001). Conclusions The combination of alanine aminotransferase, glutamyl transpeptidase, fibrinogen, platelet large cell ratio, activated partial thromboplastin time, lactate dehydrogenase, creatinine, and mean corpuscular hemoglobin concentration levels can effectively predict ICP in the first 20 weeks of gestation. These could help provide direction for earlier detection and prevention of ICP.

Activation of Platelet NLRP3 Inflammasome in Crohn's Disease.

Patients with Crohn’s disease (CD) are inclined to have platelet hyperactivity and an increased risk of intestinal micro-thrombosis. However, the mechanisms underlying platelet hyperactivity in CD are not well understood. We investigated the assembly of platelet NLRP3 inflammasome in patients with active CD and its correlation with platelet hyperactivity. In this study, Real-time PCR and western blotting analyses uncovered that ASC, NLRP3, and active caspase-1 were significantly upregulated in platelets from patients with active CD compared with healthy subjects. As revealed by flow cytometry (FCM) and ELISA analyses, the levels of interleukin-1β in both serum and isolated platelets were elevated in patients with active CD. Co-immunoprecipitation and immunofluorescence experiments revealed an increased assembly of NLRP3 inflammasome in platelets from patients with active CD. In addition, higher levels of intracellular reactive oxygen species (ROS) were observed in these platelets by FCM. Furthermore, elevated levels of platelet P-selectin exposure and fibrinogen binding were demonstrated in patients with active CD by FCM. They were positively correlated with the protein levels of NLRP3 inflammasome components. Collectively, our results indicate that the ROS-NLRP3 inflammasome-interleukin-1β axis may contribute to platelet hyperactivity in active CD.

Biomarkers of Platelet Activation and Their Prognostic Value in Patients in Sepsis Associated Coagulopathy

Introduction:Sepsis-associated disseminated intravascular coagulation (SAC) is associated with marked hemostatic changes including transient thrombocytopenia due to their enodogenous activation / consumption. Platelets are decreased in SAC and exhibit compromised function. Patients with SAC are at an increased risk of thrombosis and / or bleeding. The widespread activation of platelets contribute to vascular occlusions, fibrin deposition, multi-organ dysfunction, contributing to a two-fold increase in mortality. At this time there is no single biomarker or laboratory test that can effectively diagnose SAC; however, the International Society on Thrombosis and Hemostasis (ISTH) has introduced a scoring system based on laboratory parameters such as platelet count, clotting time, and levels of markers of thrombosis to calculate SAC severity in terms of DIC score. Measurement of activation markers that describe the interplay between inflammation and platelet function may provide insight into the pathogenesis of SAC. The purpose of this study was to measure markers of platelet function in the plasma of patients with clinically established SAC and to determine their association to disease severity and outcome.Materials and Methods:Plasma samples from 103 adult intensive care unit (ICU) patients with sepsis and suspected disseminated intravascular coagulation (DIC) were acquired from the University of Utah Hospital at the time of ICU admission as well as on days 4 and 8. Samples with relevant clinical information were transported to Loyola University Chicago under an IRB-approved protocol and stored at -80˚C prior to analysis. Plasma samples from healthy individuals (n=50) were purchased from George King Biomedical (Overland, KS). DIC scores were calculated using the ISTH scoring algorithm which incorporates platelet count, D-Dimer, INR, and fibrinogen. Patients were categorized as having no DIC, non-overt DIC, or overt DIC. Plasma levels of CD40L, von Willebrand Factor (vWF), platelet factor-4 (PF-4), and microparticles (MP) were quantified using commercially available ELISAs performed according to manufacturer instructions.Results:Markers of platelet activation were significantly elevated in patients with sepsis alone and with suspected DIC compared to normal healthy individuals on ICU day 0 (p<0.001). Levels of platelet-associated biomarkers were compared between survivors and non-survivors. PF-4 was significantly decreased in non-survivors compared to survivors (p = 0.0156). No other significant variations based on survival or based on DIC score category were observed.Since platelet count is often dramatically reduced in patients with DIC, the relationship between platelet count and levels of markers of platelet activity was also evaluated. Patients were stratified based on platelet count and levels of markers were compared between groups. CD40L, vWF, PF4, and MP showed significant variation based on platelet count, with all markers exhibiting stepwise elevation with increasing platelet count (Table 1).Conclusion: Markers of platelet activation were significantly elevated in patients with SAC compared to healthy individuals. This increase was independent of platelet count, underscoring the importance of activation processes to this disease process. Of the markers studied, PF4 showed significant difference based on DIC score or mortality, and differentiated the non-survivors compared to survivors. Levels of CD40L, vWF, PF4, and MP showed significant association with platelet count, increasing in a stepwise manner with increases in platelet count. These studies also suggest that the markers of platelet activation in SAC are primarily regulated by the number of circulating platelets and may be independent of the factors leading to their endogenous consumption. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abnormal Coagulation Function in CD19-Targeted CAR-T Therapy

Cytokine release syndrome (CRS), central nervous system (CNS) abnormalities and B-cell aplasia have been widely reported in recent clinical trials for chimeric antigen receptor T-cell (CAR-T) therapy. In our study, we also note that patients who received CAR-T therapy were easily to develop coagulopathy, even disseminated intravascular coagulation (DIC), which increased the risk of treatment related mortality (TRM) and required timely intervention. Therefore, it is important to pay attention to the symptom and find out the way to improve it.In our phase-1 clinical trial (NCT02965092) for B-cell acute lymphoblastic leukemia (B-ALL), we dynamically monitored the coagulation indicators and treated coagulopathy. Twenty-five patients (median age 28, [10-61]) were recruited into the trial and they underwent leukapheresis on day -11. Then they were subjected to the lymphodepleting regimens (fludarabine + cyclophosphamide, n=22; fludarabine, n=2; cyclophosphamide, n=1) on day -7~-5. Finally, they received the infusions of anti-CD19 CAR-T cells at a median dose of 4.5×106/kg (3.2-8.3×106/kg) on day 0~2. Excluding 3 patients who quitted from the trial for rapid progression of disease, and 3 patients who have not reached one-month follow-up time point yet, the overall one-month response rate for the evaluable 22 patients was 86.36%. Up to date, the longest leukemia free survival (LFS) was 16 months (median 5 months, [1-16]). As for side effects, 12 patients developed grade 2-3 CRS, 2 patients suffered from CNS symptoms, and no patients experienced serious infections. Besides, the obvious coagulopathy caught our attention. Eleven patients (11/22, 50%) developed coagulation dysfunction, manifested as petechiae and abnormal laboratory indicators (Table 1), such as deceased platelet count and fibrinogen, increased D-Dimer and fibrin degradation products, prolongations of activated partial thromboplastin time, prothrombin time and thrombin time. According to Chinese DIC Scoring System (CDSS), 5 of these 11 patients should be diagnosed as DIC (Table 2).Furthermore, we collected patients' plasma to measure the concentrations of tissue factor (TF) and platelet endothelial cell adhesion molecular-1 (PECAM-1), which are 2 vascular endothelial molecules that may play important roles in inflammation and coagulation. It was observed that coagulation disorders were more common in patients who developed severe CRS. Here we present the experiment results of 3 patients who experienced severe CRS and DIC at the same time. As shown in Figure 1, when the concentration of interleukin 6 rose and indicated CRS, the coagulopathy happened. At the same time, the TF concentration rose and PECAM-1 concentration dropped. Taken together, these data indicate that there is a relationship between CRS and DIC. We hypothesize that the cytokines released from inflammatory cells during CRS could cause dysfunction of endothelial cells and destroy the integrity of endothelial barrier, thus resulting in coagulopathy. However, the mechanism remains poorly understood, more in vivo experiments should be conducted to identify the mechanism, which may suggest novel therapeutic targets for coagulopathy.In conclusion, our data demonstrate that coagulation disorders are common side effects in CAR-T therapy, and suggest that TF and PECAM-1 are critical parameters in the pathogenesis of CRS-related coagulopathy. Investigators should pay more attention to coagulopathy and take preventive measures to avoid DIC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomarkers of Endothelial Function in Sepsis-Associated Disseminated Intravascular Coagulation

Introduction: Sepsis-associated disseminated intravascular coagulation (DIC) is a complex clinical syndrome with high mortality. The pathogenesis of DIC involves significant interactions between multiple pathophysiologic processes including coagulation, inflammation, infection response, platelets and endothelial dysfunction. The role of the endothelium in DIC is multifaceted and may contribute significantly to its severity and outcome. In sepsis, increased inflammation can lead to activation of the endothelium and the development of a procoagulant phenotype, characterized in part by elevated levels of cell surface tissue factor (TF). The endothelial response to inflammation is also characterized by the production of other factors that may be descriptive of the status of the endothelium, including endocan. The endothelium is also largely responsible for the production of endogenous anticoagulants, such as Tissue Factor Pathway Inhibitor (TFPI) and Protein C. The derangement of the endogenous anticoagulant system is critical to the pathogenesis of DIC and may also be reflective of the state of the endothelium. Investigation of factors that may prove damaging to the endothelium, including extracellular nuclear material such as nucleosomes and the protein High Mobility Group Box 1 (HMGB-1) may also provide insight into endothelial status and associated patient prognosis. The purpose of this study was to quantify measures of endothelial function, including markers of activation (TF and endocan), endogenous anticoagulants (TFPI and Protein C), and damage-associated factors (nucleosomes and HMGB-1) in the plasma of patients with sepsis and DIC and to determine the relationship of these factors with severity of illness and outcome.Materials and Methods: Citrated plasma samples were prospectively collected from 103 patients with sepsis and suspected DIC at ICU admission (day 0) and again serially on ICU days 4 and 8 under an IRB approved protocol. DIC score was determined using the ISTH scoring algorithm (e.g. platelet count, PT/INR, fibrinogen, and D-Dimer). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland, KS). Plasma levels of endocan, TF, TFPI, nucleosomes, and HMGB-1 were evaluated using commercially available ELISA kits. Protein C levels were measured using a functional clot-based assay.Results: In the DIC patients, endocan, TF, TFPI, nucleosomes, and HMGB-1 were significantly elevated compared to normal on ICU days 0, 4, and 8. Protein C was significantly reduced compared to normal at all time points.Patients were stratified into groups of sepsis and DIC based on the ISTH DIC score, and levels of markers were compared between groups. Markers reflective of overall endothelial activation (endocan and TF) showed no variation based on DIC score. Protein C showed significant variation based on DIC score, with a marked drop in Protein C levels with increasing severity of illness; however, TFPI showed no variation. Increasing levels of factors associated with endothelial damage (nucleosomes and HMGB-1) were observed with increasing severity of illness. Results are shown in Table 1.Differences in biomarker levels between survivors and non-survivors were also evaluated. Endocan (p = 0.0252) and HMGB-1 (p = 0.0306) showed significant increases in non-survivors compared to survivors while Protein C showed a significant reduction (p = 0.0093) in non-survivors compared to survivors. TF, TFPI, and nucleosomes showed no variation based on survival.Conclusion: Overall, levels of markers of endothelial activation, endogenous anticoagulants, and factors contributing to endothelial damage were elevated in the plasma of patients with sepsis-associated DIC compared to the healthy population. In particular, factors associated with endothelial damage, namely the toxic nuclear factors nucleosomes and HMGB-1, showed elevated levels with increasing severity of coagulopathy, suggesting that the endothelial damage caused by these factors may contribute to the development of coagulation dysfunction. Levels of endocan, Protein C, and HMGB-1 also showed significant association with mortality, underscoring the relevance of all aspects of endothelial physiology to patient outcome in DIC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIa.

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

Laboratory testing for suspected COVID-19 vaccine-induced (immune) thrombotic thrombocytopenia.

COVID‐19 (coronavirus disease 2019) represents a pandemic, and several vaccines have been produced to prevent infection and/or severe sequelae associated with SARS‐CoV‐2 (severe acute respiratory syndrome coronavirus 2) infection. There have been several reports of infrequent post vaccine associated thrombotic events, in particular for adenovirus‐based vaccines. These have variously been termed VIPIT (vaccine‐induced prothrombotic immune thrombocytopenia), VITT (vaccine‐induced [immune] thrombotic thrombocytopenia), VATT (vaccine‐associated [immune] thrombotic thrombocytopenia), and TTS (thrombosis with thrombocytopenia syndrome). In this report, the laboratory test processes, as utilised to assess suspected VITT, are reviewed. In published reports to date, there are notable similarities and divergences in testing approaches, potentially leading to identification of slightly disparate patient cohorts. The key to appropriate identification/exclusion of VITT, and potential differentiation from heparin‐induced thrombocytopenia with thrombosis (HITT), is identification of potentially differential test patterns. In summary, testing typically comprises platelet counts, D‐dimer, fibrinogen, and various immunological and functional assays for platelet factor 4 (PF4) antibodies. In suspected VITT, there is a generally highly elevated level of D‐dimer, thrombocytopenia, and PF4 antibodies can be identified by ELISA‐based assays, but not by other immunological assays typically positive in HITT. In addition, in some functional platelet activation assays, standard doses of heparin have been identified to inhibit activation in suspected VITT, but they tend to augment activation in HITT. Conversely, it is also important to not over‐diagnose VITT, given that not all cases of thrombosis post vaccination will have an immune basis and not all PF4‐ELISA positive patients will be VITT.

Ultrasound‐Responsive Nanopeptisomes Enable Synchronous Spatial Imaging and Inhibition of Clot Growth in Deep Vein Thrombosis

Deep vein thrombosis (DVT) is a life-threatening blood clotting condition that, if undetected, can cause deadly pulmonary embolisms. Critical to its clinical management is the ability to rapidly detect, monitor, and treat thrombosis. However, current diagnostic imaging modalities lack the resolution required to precisely localize vessel occlusions and enable clot monitoring in real time. Here, we rationally design fibrinogen-mimicking fluoropeptide nanoemulsions, or nanopeptisomes (NPeps), that allow contrast-enhanced ultrasound imaging of thrombi and synchronous inhibition of clot growth. The theranostic duality of NPeps is imparted via their intrinsic binding to integrins overexpressed on platelets activated during coagulation. The platelet-bound nanoemulsions can be vaporized and oscillate in an applied acoustic field to enable contrast-enhanced Doppler ultrasound detection of thrombi. Concurrently, nanoemulsions bound to platelets competitively inhibit secondary platelet-fibrinogen binding to disrupt further clot growth. Continued development of this synchronous theranostic platform may open new opportunities for image-guided, non-invasive, interventions for DVT and other vascular diseases.

Fabrication of Citric Acid/RGD Multilayers on Mg‐Zn‐Y‐Nd Alloy via Layer‐by‐Layer Self‐Assembly for Promoting Surface Biocompatibility

AbstractMagnesium (Mg) alloy, an important biodegradable material widely investigated on cardiovascular stent, is restricted for further application due to some limited functions, such as the excessively rapid degradation rate, poor hemocompatibility, and delayed re‐endothelialization. To overcome these shortcomings, surface modification with biomolecules has attracted much attention. In this study, the multifunctional coatings of citric acid (CA) and Arg‐Gly‐Asp (RGD) via layer‐by‐layer self‐assembly are immobilized onto the polydopamine (PDA) deposited surface which is passivated with hydrofluoric acid (HF) solution before. The basic characterization results suggest that PDA/(CA‐RGD)2 coating exhibits a rich‐in‐amine and hydrophilic surface. The corrosion current density of PDA/(CA‐RGD)2 exposed to simulated body fluid is reduced by 95.65% from 1.586 × 10−4 to 6.894 × 10−6 A cm−2 under the protection of the dense MgF2 layer and multifunctional coatings. Besides, inhibition on platelet adhesion, hemolysis rates, and fibrinogen adsorption also prove the enhanced hemocompatibility of the PDA/(CA‐RGD)2 coatings. Meanwhile, the human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) culture results jointly prove the excellent endothelialization performance of functionalized samples. In conclusion, the above mentioned results reveal that the functionalized surface of PDA/(CA‐RGD)2 displays better overall performance, indicating a preferable application for cardiovascular stents.

Hypocoagulable Tendency on Thromboelastometry Associated With Severity and Anticoagulation Timing in Pediatric Septic Shock: A Prospective Observational Study.

Objective: To identify whether coagulation profiles using thromboelastometry are associated with outcomes in pediatric septic shock. The primary outcomes were the development of disseminated intravascular coagulation (DIC) and the severity of the pediatric intensive care unit (PICU) existing scoring systems, while the secondary outcome was hospital mortality. This study aimed to contribute to current findings of the limitations of conventional tests in determining the optimal timing of anticoagulation in sepsis.Design: A prospective, observational study conducted between August 2019 and August 2020.Setting: PICU at a pediatric tertiary hospital in Hanoi, Vietnam.Patients: Fifty-five pediatric patients who met the septic shock criteria were enrolled.Measurements and Main Results: Fifty-five patients with septic shock were recruited. At the time of diagnosis, thromboelastometry revealed normocoagulability, hypercoagulability, and hypocoagulability in 29, 29, and 42% of the patients, respectively (p > 0.05); however, most patients in the overt DIC and non-survival groups progressed to hypocoagulability (82 and 64%, respectively). The overt DIC, PELOD-2 > 8, PRISM-III > 11, and non-survival group had a significant hypocoagulable tendency according to thromboelastometry parameters [prolonged clotting time (CT) and clot formation time (CFT); and reduced α-angle (α), maximum clot firmness (MCF), thrombodynamic potential index (TPI)] compared to the non-overt DIC, PELOD-2 ≤ 8, PRISM-III score ≤ 11 and survival group (p < 0.05). Conventional parameters between the normocoagulable and hypercoagulable groups were not different (p > 0.05). Hypocoagulability was characterized by lower platelet count and fibrinogen level, higher prolonged prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (APTT), and higher D-dimer level than in hypercoagulability (p < 0.05). Hypocoagulable tendency on thromboelastometry had a higher hazard at a PT > 16.1 s [area under the curve (AUC) = 0.747, odds ratio (OR) = 10.5, p = 0.002], INR > 1.4 (AUC = 0.754, OR = 6.9, p = 0.001), fibrinogen <3.3 g/L (AUC = 0.728, OR = 9.9, p = 0.004), and D-dimer > 3,863 ng/mL (AUC = 0.728, OR = 6.7, p = 0.004).Conclusions: Hypocoagulable tendency using thromboelastometry is associated with the severity of septic shock. Conventional coagulation tests may fail to detect hypercoagulability, which is crucial in determining anticoagulation timing.