Whole Blood Procoagulant Platelet Flow Cytometry Protocol for Heparin-Induced Thrombocytopenia (HIT) and Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Testing.

Heparin-induced thrombocytopenia (HIT) is a well-characterized, iatrogenic complication of heparin anticoagulation with significant morbidity. In contrast, vaccine-induced immune thrombotic thrombocytopenia (VITT) is a recently recognized severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria, AstraZeneca) and Ad26.COV2.S (Janssen, Johnson & Johnson) vaccines against COVID-19. The diagnosis of HIT and VITT involve laboratory testing for antiplatelet antibodies by immunoassays followed by confirmation by functional assays to detect platelet-activating antibodies. Functional assays are critical to detect pathological antibodies due to the varying sensitivity and specificity of immunoassays. This chapter presents a protocol for a novel whole blood flow cytometry-based assay to detect procoagulant platelets in healthy donor blood in response to plasma from patients suspected of HIT or VITT. A method to identify suitable healthy donors for HIT and VITT testing is also described.

Long COVID-19 syndrome: Association of cardiopulmonary impairment with a persistent platelet activation

Background: A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: This condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID- 19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Purpose(s): To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients. Method(s): 6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows- CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry;pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Result(s): Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2-2.9] vs 2.4% [1.6-5.7]) and 2-fold (217/mul [137-275] vs 435/mul [275-633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3-13.5] vs 11.7% [5.2-18.9]) and PLA formation (35.5% [27.4- 46.8] vs 67.7% [45.7-85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=-0.423;p=0.02). Conclusion(s): These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients.

Beware of the Unexpected: Acute Coronary Thrombosis Following the Mildly Symptomatic COVID-19 Infection and Two Different Therapeutic Options

COVID-19 infection was declared a pandemic in March 2020 and is responsible for high morbidity and mortality around the globe. Although the most common clinical presentation is respiratory, cardiac complications are common and create new challenges for healthcare. We discuss two mildly symptomatic outpatients with COVID-19 presenting with acute ST-elevation myocardial infarction (STEMI) and high coronary thrombus burden without atherosclerotic plaque. Our report describes two different therapeutic approaches that illustrate the challenges encountered in the management of disseminated coronary thrombosis in patients with STEMI and COVID-19. Clinicians should be aware of the high pro-coagulant state caused by COVID-19, even in mildly symptomatic outpatients, and aggressive pharmacological therapy may be an effective alternative treatment option to percutaneous coronary intervention.Copyright © 2023, CKS.

Establishment of an in vitro assay to detect antibody-mediated procoagulant platelet formation on a single cell level in real time

Introduction Procoagulant platelets (PLTs), a subpopulation of PLTs that is characterized by increased externalization of phosphatidylserine (PS), are increasingly identified to promote a prothrombotic environment in different diseases. Recently we observed that procoagulant PLT formation can be induced via engagement of immune receptor Fc-gamma-RIIA by COVID-19, VITT and HIT patient immunoglobulin subclass G (IgG) antibodies (Abs). Here, Fc-gamma- RIIA engagement by patient Abs resulted in significant formation of procoagulant PLTs and loss of mitochondrial potential that was associated with high thrombin formation as well as increased thrombus formation. In the cur- rent study, we aim to establish a PLT adhesion assay that allows investigation of PLT mitochondria during procoagulant PLT formation. Method PLTs were spread on human serum albumin, fibrinogen or collagen precoated glass slides. Adhesion and subsequent shape change of PLTs as well as procoagulant PLT formation were investigated in real time using immune fluorescence microscopy. For the detection of PLT shape change, mitochondrial dynamics and PS externalization, PLTs were double stained with MitoTracker green, a mitochondrial dye that binds to free thiol groups of cysteine residues in the mitochondrial membrane, and Annexin-V, respectively. For the visualization of mitochondrial release from PLTs intracellular compartment, a monoclonal Ab that binds to a subunit of the translocase of the outer membrane (TOM) complex on the mitochondrial membrane, namely TOM22, was used. Results During the observation period, a subgroup of PLTs that was spread on collagen became procoagulant as determined by an increased binding of Annexin- V on the PLT surface. Contrary, these changes were nearly absent in PLTs that adhered to fibrinogen (percentage [ %] of Annexin-V positive cells: 19.80 +/- 3.42 % vs. 1.92 +/- 0.62 %, p value 0.0357). Interestingly, procoagulant PLT formation was associated with a significant loss of MitoTracker green signal in PLTs while it remained constant in non-procoagulant PLTs attached on both extracellular matrix coatings. Loss of MitoTracker green signal was associated with translocation of mitochondrial proteins from intracellular to extracellular, as a higher count of TOM22 Ab-positive labelled structures, most likely extracellular mitochondria were detected on collagen but not on fibrinogen coated glass slides. Conclusion Our findings indicate, that the formation of procoagulant PLTs is associated with dramatic changes of the mitochondrial integrity in PLTs. Further attempts, that investigate the potential pathophysiological role of PLT mitochondrial release in Ab-mediated prothrombotic disorders may contribute to a further understanding of the role of PLT mitochondria in these complex diseases.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

COVID-19-ASSOCIATED THROMBOTIC THROMBOCYTOPENIA PURPURA (TTP)

SESSION TITLE: COVID-Related Critical Care Cases SESSION TYPE: Case Reports PRESENTED ON: 10/19/2022 11:15 am - 12:15 pm INTRODUCTION: Thrombotic thrombocytopenic purpura (TTP) is a rare medical emergency with mortality rates reported to be as high as 90% if untreated. We report a case of severe TTP in an immunocompetent patient diagnosed with COVID 19 infection. CASE PRESENTATION: A 34-year-old morbidly obese female not vaccinated for COVID presented to PCP 2 weeks prior with complaints of fatigue. CBC showed Hb 12.2, platelet count 108 (baseline > 200), and covid resulted positive. The patient was reluctant for further workup at the time. After 2 weeks she felt short of breath prompting ICU admission for high flow oxygen with blood work showing hemoglobin of 8.8, platelet count of 11, Reticulocyte count 3.9%, LDH 763, fibrinogen 639, schistocytes on peripheral smear, MCV < 90, INR<1.5, Creatinine 1 giving her a PLASMIC Score of 7. For concerns of TTP, she was urgently started on plasmapheresis, prednisone 1 mg/kg, and remdesivir for COVID. ADAMTS13 was sent prior to the initiation of plasmapheresis resulted at 5% indicating severe deficiency and a high risk of relapse. She underwent 3 cycles of plasma exchange and was also started on Caplacizumab. Rituximab was not started in the setting of active COVID infection and negative COVID IgG. Repeat ADAMTS13 level at 2-week interval increased to 43%. The patient was discharged on steroids and completed 1 month of Caplacizumab. On outpatient follow up the patient was asymptomatic but repeat ADAMTS13 declined to 16%. COVID IgG now resulted positive, and she was started on Rituximab. Ultimately patient was treated with 4 months of steroids and 4 doses of weekly Rituximab with the final two ADAMTS 13 levels normalized above 100. DISCUSSION: TTP is caused by decreased activity of the plasma metalloproteinase ADAMTS 13, the key enzyme involved in the cleavage of ultra-large von Willebrand Factor (vWF) multimers into smaller less procoagulant multimers. It is reported that COVID-19 infection is associated with almost a five-fold increase in vWF levels which the body's ADAMTS-13 enzyme activity cannot adequately regulate, resulting in an excess of unchecked ultra-large vWF, diffuse microthrombi, and systemic ischemia. The presentation of this disease is often characterized by the pentad of fever, thrombocytopenia, hemolytic anemia, renal dysfunction, and neurologic dysfunction. However, the full pentad is often not present in many patients Plasma exchange and immunosuppression are the mainstays of treatment for TTP. A high index of suspicion is required for a timely diagnosis. Early diagnosis is crucial as without treatment TTP is associated with a high mortality rate. CONCLUSIONS: Health care providers should be aware of this life-threatening complication of COVID-19 so that prompt and appropriate interventions can be undertaken if it is suspected or confirmed. Rituximab should be delayed until the acute COVID-19 infection has cleared, and neutralizing antibodies have been produced. Reference #1: Hindilerden F, Yonal-Hindilerden I, Akar E, Kart-Yasar K. Covid-19 associated autoimmune thrombotic thrombocytopenic purpura: Report of a case. Thromb Res. 2020;195:136-138. doi:10.1016/j.thromres.2020.07.005 DISCLOSURES: No relevant relationships by Hanish Jain No relevant relationships by Dragos Manta No relevant relationships by Parth Sampat No relevant relationships by Garima Singh No relevant relationships by Simant Thapa

A PATIENT WITH COVID-19 PNEUMONIA, PULMONARY EMBOLISM, AND OSA

SESSION TITLE: Global Case Reports in Critical Care SESSION TYPE: Global Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 01:45 pm INTRODUCTION: Thrombotic complications in patients diagnosed with COVID-19 pneumonia are emerging as an important and significant morbidity and mortality burden, with overwhelming inflammation, hypoxia, immobilization, and diffuse intravascular coagulation among possible causes of a procoagulant state (1). Obstructive sleep apnea (OSA), with intermittent arterial oxygen desaturation, may in its turn contribute to a procoagulant state, causing hemodynamic alterations as polycythemia and sluggish blood flow (2). Here we report on a case of sudden and massive non-lethal pulmonary thromboembolism (PTE) in a patient with COVID-19 severe pneumonia, for whom OSA was suspected and documented as a possible concurrent mechanism of thromboembolic complication during follow-up. CASE PRESENTATION: A 55-year-old male non-smoker obese (BMI 33 Kg/m2) was admitted to our hospital after 9 days of fever. In the Emergency Room, a chest HRCT scan showed bilateral diffuse ground glass opacities. He was treated with subcutaneous Tocilizumab (324 mg) single shot, Remdesivir (200 mg/day for first day and 100/daily for further 4 days), methyl-prednisolone 40 mg/daily, Enoxaparin 6000 UI/twice daily, azithromycin 500 mg/daily, high flow nasal cannula oxygen (50 L/min, TC 34°C, FiO2 35%) for moderate acute respiratory failure due to COVID-19 pneumonia (pO2: 58 mmHg, PCO2 34 mmHg pH 7.50, P/F 275). After 10 days, patient's clinical conditions worsened, needing non-invasive respiratory support;D-dimer increased abruptly, rising to 10 ng/mL, with findings consistent with PTE at a computed tomographic angiography (CTA, Fig 1). The patient was successfully treated with 10 mg/daily subcutaneous fondaparinux for 12 days, while assisted in the Intensive Care Unit, being discharged home in room air shortly later with oral anticoagulants. At the 3-month follow-up visit, OSA was suspected due to reported excessive daytime sleepiness and weakness, snoring, disturbed night sleep, morning headache in the last 4 years. The patient underwent a home sleep apnea test (HSAT) overnight. Test results revealed an AHI of 50 events/h, with several prolonged episodes of obstructive sleep apnea (307 apnea and hypopnea (A+H) events, 70 obstructive apnea and 233 hypopnea events, with a mean duration of 10% and an average arterial saturation of 93% (Fig. 2). He was adapted to CPAP therapy, with benefit and good correction of polygraphic indexes. DISCUSSION: The pathogenetic mechanisms of COVID 19 and OSA could have played a synergistic effect on endothelial damage, thus increasing the risk of thromboembolism. CONCLUSIONS: The presence of underdiagnosed comorbidities may well worsen the clinical course and complication of COVID-19;an earlier diagnosis of OSA is a prerequisite for timely treatment and, potentially, improved long-term clinical outcomes. Reference #1: Suh YJ, et al. Pulmonary embolism and deep vein thrombosis in COVID 19: a systematic review and meta-analysis. Radiology 2021;298 (2): E70-E80. Reference #2: Alfonso-Fernandez A., Garcia Surquia A., de la Pena M. OSA is a risk factor for recurrent VTE Chest. 2016;150 (6): 1291-1301. DISCLOSURES: no disclosure on file for Antonietta Esposito;no disclosure on file for Antonella Frattari;no disclosure on file for Giustino Parruti;no disclosure on file for Giorgia Patrizio;no disclosure on file for Pierpaolo Prosperi;no disclosure on file for Giorgia Rapacchiale;No relevant relationships by ANTONELLA SPACONE no disclosure on file for Giacomo Zuccarini;

FEATURES OF THE PARAMETERS OF RED BLOOD CELLS AND HEMOSTASIS IN PATIENTS WITH STROKES ASSOCIATED WITH CORONAVIRUS INFECTION

Objective: The purpose is to identify the peculiarities of the parameters of red blood cells (RBC) and hemostasis in patients with strokes associated with coronavirus infection. Design and method: A total of 124 patients (48.5 + 1.9 years) with impairments of cerebral circulation due to COVID-19 (confirmed by positive PCR test) had been examined. Among them, 74 patients had ischemic stroke, 25- transient ischaemic attack, 17- intracerebral hemorrhage, 8- subarachnoid hemorrhage. The parameters of hemostasis were measured by standard methods, electrical, viscoelastic parameters of RBC - by dielectrophoresis. Results: 71 patients (the 1st group) showed signs of intravascular coagulation and thrombosis: accelerated platelet-leukocyte aggregation, increased levels of coagulation products, reduced fibrinolysis activity (p = 0.001-0.04). The levels of D-dimer, fibrinogen, ESR, platelet count were higher in this group compared to the second one (p < 0.01). A moderate increase of RBC summarized rigidity, viscosity was noted. The level of RBC hemolysis was associated with platelet count (r = 0.735,p = 0.03), D-dimer (r = 0.482, p < 0.05), fibrinogen level (r = 0.374, p = 0.04). In 2nd group (53 persons), the markers of thrombosis had moderate deviations. Sharply reduced RBC deformability with increased summarized rigidity, viscosity was dominant coupled with the background of high electrical conductivity of cell membranes compared to the indicators in the 1st group (p < 0.01). There was a decrease of membrane capacity, surface charge, cell dipole moment, polarizability than those in the 1st group (p = 0.0001-0.05). A sharp decrease of RBC deformability creates obstacles to overcoming small-diameter capillaries, leading to violations of microcirculatory blood flow. RBC deformability was associated with levels of ferritin (r = 0.451, p = 0.02), HbA1c (r = 0.480, p = 0.03), uric acid (r = -0.371, p < 0.05), LDL cholesterol (r = 0.461, p = 0.02). Incubation of blood samples in vitro for 10 min with riboflavin, nicotinamide, inosine, which ensures RBC energy metabolism, restored the reduced RBC deformability (p < 0.01), altered cell morphology (p = 0.04), decreased RBC aggregation (p < 0.001). Conclusions: The revealed features of parameters of RBC hemostasis in stroke patients with coronavirus infection are associated with two independent pathogenetic mechanisms: thrombotic and hemorheologic. The thrombotic variant is due to procoagulant state and an activity of inflammation. The hemorheologic variant is caused by decrease of RBC energy metabolism, activity of enzymes.

Platelets genetically modified with mRNA-lipid nanoparticles are functionally similar to clinically transfused platelets in vitro

Background: Platelet transfusions are an essential treatment for attenuating bleeding but are often ineffective in cases of intractable haemorrhage. Although anucleate, mature platelets synthesize protein de novo, making them amenable to mRNA gene therapy;however, there remains to be an effective transfection technique. Advancements in lipid nanoparticle technology has enabled leading COVID vaccines and is an efficient method to deliver nucleic acids into target cells. Recently, we developed a LNP approach to successfully express exogenous protein in platelets [unpublished data], a first step towards demonstrating that donor platelet coagulability can be engineered. However, the effects of LNP treatment on platelet function has yet to be investigated. Aims: To determine whether LNP-treated donor platelets are functionally similar, or better, in vitro, than platelets currently transfused clinically as a next step to establish LNP engineered platelets as a new cell therapy. Methods: The hemostatic profiles of LNP-treated and clinical donor platelets were assessed using an adapted rotational thromboelastometry model of dilutional coagulopathy. LNP-treated platelets were also stimulated with conventional platelet agonists to test if responsiveness is similar, or better than clinical platelets using flow cytometry. Results: LNP-treated platelets have a comparable hemostatic profile to clinically transfused platelets and significantly improved clotting dynamics when spiked into hemodiluted whole blood in an in vitro transfusion simulation. LNP-treated platelets also respond comparably, and in some cases more potently to agonist simulation compared to untreated platelets as indicated by similar p-selectin surface presentation. Summary/Conclusions: LNPs are an effective way to deliver exogenous nucleic acids into platelets;they do not significantly change platelet coagulability or responsiveness to agonists. LNP platelet engineering is a promising new approach to load platelets with procoagulant protein to enhance their function.

Immune-Mediated Platelet Activation in COVID-19 and Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Both qualitative and quantitative platelet abnormalities are common in patients with coronavirus disease 2019 (COVID-19) and they correlate with clinical severity and mortality. Activated platelets contribute to the prothrombotic state in COVID-19 patients. Several groups have shown immune-mediated activation of platelets in critically ill COVID-19 patients. Vaccine-induced immune thrombotic thrombocytopenia is an autoimmune condition characterized by thrombocytopenia and life-threatening thrombotic events in the arterial and venous circulation. Although the initial trigger has yet to be determined, activation of platelets by immune complexes through Fc gamma RIIA results in platelet consumption and thrombosis. A better understanding of platelet activation in COVID-19 as well as in vaccine-induced thrombotic complications will have therapeutic implications. In this review, we focused on the role of immune-mediated platelet activation in thrombotic complications during COVID-19 infection and vaccine-induced immune thrombotic thrombocytopenia.

Procoagulant Microvesicles in COVID-19 Patients: Possible Modulators of Inflammation and Prothrombotic Tendency.

Background: The hypercoagulability and thrombotic tendency in coronavirus disease 2019 (COVID-19) is multifactorial, driven mainly by inflammation, and endothelial dysfunction. Elevated levels of procoagulant microvesicles (MVs) and tissue factor-bearing microvesicles (TF-bearing MVs) have been observed in many diseases with thrombotic tendency. The current study aimed to measure the levels of procoagulant MVs and TF-bearing MVs in patients with COVID-19 and healthy controls and to correlate their levels with platelet counts, D-Dimer levels, and other proposed calculated inflammatory markers. Materials and Methods: Forty ICU-admitted patients with COVID-19 and 37 healthy controls were recruited in the study. Levels of procoagulant MVs and TF-bearing MVs in the plasma of the study population were measured using enzyme linked immunosorbent assay. Results: COVID-19 patients had significantly elevated levels of procoagulant MVs and TF-bearing MVs as compared with healthy controls (P<0.001). Procoagulant MVs significantly correlated with TF-bearing MVs, D-dimer levels, and platelet count, but not with calculated inflammatory markers (neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, and platelet/neutrophil ratio). Conclusion: Elevated levels of procoagulant MVs and TF-bearing MVs in patients with COVID-19 are suggested to be (i) early potential markers to predict the severity of COVID-19 (ii) a novel circulatory biomarker to evaluate the procoagulant activity and severity of COVID-19.

In covid-19 infection, plasma extra cellular vesicle tissue factor activity does not correlate with d-dimer levels

Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation. Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation. Methods: Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis. Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization;n=37);intensive care (ICU;n=16);mechanical ventilation (Ventilation;n=22);or fatality (Deceased;n=22). Result: Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin;pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity. Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:<0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies. Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases.

Advanced cell therapy with low tissue factor loaded product NestaCell® does not confer thrombogenic risk for critically ill COVID-19 heparin-treated patients.

Since the COVID-19 pandemic started, mesenchymal stromal cells (MSC) appeared as a therapeutic option to reduce the over-activated inflammatory response and promote recovery of lung damage. Most clinical studies use intravenous injection for MSC delivery, raising several concerns of thrombogenic risk due to MSC procoagulant activity (PCA) linked to the expression of tissue factor (TF/CD142). This is the first study that demonstrated procoagulant activity of TF+ human immature dental pulp stromal cells (hIDPSC, NestaCell® product) with the percentage of TF+ cells varied from 0.2% to 63.9% in plasma of healthy donors and COVID-19 heparin-treated patients. Thrombogenic risk of TF+ hIDPSCs was evaluated by rotational thromboelastometry (in vitro) and in critically ill COVID-19 patients (clinical trial). We showed that the thromboelastography is not enough to predict the risk of TF+ MSC therapies. Using TF-negative HUVEC cells, we demonstrated that TF is not a unique factor responsible for the cell's procoagulant activity. However, heparin treatment minimizes MSC procoagulant (in vitro). We also showed that the intravenous infusion of hIDPSCs with prophylactic enoxaparin administration in moderate to critically ill COVID-19 patients did not change the values of D-dimer, neither in the PT and PTT times. Our COVID-19 clinical study measured and selected the therapeutic cells with low TF (less than 25% of TF+ hIDPSCs). Our data indicate that the concomitant administration of enoxaparin and low TF-loaded is safe even for critically ill COVID-19 patients.

AN INTERESTING CASE OF CMV MYOPERICARDITIS IN AN IMMUNOCOMPETENT PATIENT

Background: Cytomegalovirus (CMV) is known to cause symptomatic disease in immunocompromised individuals. We present a rare case of CMV myopericarditis with findings of acute pulmonary embolism (PE) thought to be from the procoagulant effect of CMV in an immunocompetent middle-aged female. Case: 55-year-old female who presented to the ED for chest pain. Found to have non-specific EKG changes in V4-V6, troponin and COVID negative. TTE with EF 68% and pericardial effusion. ESR and CRP were elevated. Started on treatment for viral myopericarditis with colchicine, unfortunately developed diarrhea and was switched to high dose aspirin. CT coronary angiogram was performed to rule out ischemia. Calcium score of 0 and non-obstructive CAD in the LAD. CT also showed a new acute PE with no RV strain and she was started on Xarelto for anticoagulation. Infectious work up was performed, serum CMV IgM positive and CMV DNA of 18,205 copies. Oral valganciclovir was started for a total 21-day course for CMV myopericarditis. Patient was discharged with improvement of symptoms and plan for follow up with infectious diseases and cardiology for follow-up cardiac MRI. Decision-making: Symptomatic CMV infection is common in immunocompromised individuals. Immunocompetent patients that present with pulmonary embolism are far fewer. Cardiovascular complications have only been mentioned a handful of times in literature. In addition, and to the best of our knowledge, this is only the second known description of a patient to experience both myo-pericarditis and pulmonary embolism simultaneously from a symptomatic CMV infection. Conclusion: It is important for clinicians to be aware of this rare presentation of CMV, adding this to the differential diagnosis when an immunocompetent patient presents with symptoms concerning for viral pericarditis and/or evidence of pulmonary embolus.

Dermatological Manifestations in Patients with COVID-19 Pneumonia in Veracruz, Mexico

Background. A large number of viral infections are characterized by the presence of cutaneous manifestations. Multiple dermatological manifestations have been observed in patients with COVID-19. Dermatological lesions in patients infected by SARS-CoV-2 such as livedo reticularis, rash and vascular lesions may represent manifestations of secondary phenomena such as paraviral rashes or by participation of the innate or adaptive immune system that cause vasodilation, vascular leakage or procoagulant effects Methods. Descriptive and observational study, adult patients with COVID-19 pneumonia were selected, confirmed by RT-PCR and chest CT. General symptoms, hematic cytometry results, pneumonia severity, prognosis as well as dermatological manifestations are characterized. Results. 100 patients were entered into the study, with an average age of 49.4 years, 54% male. The general symptoms with the highest incidence were: fever, cough and dyspnea characteristic of SARS-CoV-2 infection, followed by chest pain, headache, anosmia and dysgeusia. The main alteration of the hemogram was lymphopenia, no leukopenia or plaquetopenia was demonstrated. 54% of those affected had mild pneumonia, the rest severe pneumonia. 75% progressed towards improvement and 25% died. Among the dermatological manifestations identified, all occurred in cases with severe pneumonia, the one with the highest incidence was the morbilliform viral exanthema in 18%, the presence of diffuse partial alopecia in 7% as well as manifestations of lividity and maceration in 1%. Regarding alopecia, in 6% it was reversible androgenetic alopecia, having manifested during the acute stage of pneumonia (all men), in 1% it presented alopecia areata (male) that has been persistent beyond the acute phase and in frank recovery Demographic and clinical variables Conclusion. The incidence of dermatological manifestations is low in this study population, the most frequent being the morbilliform viral exanthema expected in a virus, however they present manifestations of low incidence such as reversible androgenetic alopecia associated with severity of the disease, a finding that has been documented recently as a manifestation associated with COVID-19.

Massive Haemoperitoneum Postovulation: A Rare Complication of Unmonitored Anticoagulant Therapy

Derangements in coagulation profile due to on-going anticoagulant medication can complicate the most primitive physiology in a woman, ovulation. Unmonitored anticoagulant therapy can lead to an array of complications, one of which is intraperitoneal haemorrhage secondary to numerous medical and surgical conditions. The resultant haemoperitoneum can cause sudden hypovolemic shock, especially in a patient with compromised cardiovascular status. This report narrates the case of a 30 years old nulliparous female patient on anticoagulant therapy, operated for multiple cardiac defects and developed massive haemoperitoneum as a consequence of ovulation. The patient presented in shock to the Emergency Department with unstable haemodynamic status. A multi-disciplinary approach to the case converged on ruptured functional ovarian cyst as a working diagnosis. Conservative management of haemoperitoneum was decided upon, whereby correcting shock and providing pro-coagulant therapy along with blood products was the main line of management. All efforts failed soon after when the cyst wall ruptured catastrophically, leading to collapse of the general condition of the patient. Surgical approach was undertaken and a ruptured ovarian cyst wall was identified to be the cause. The cyst wall was subsequently repaired electrosurgically and the patient recovered well with an uneventful postoperative period. However, the characteristic feature of this case is the lack of follow-up due to lockdown restrictions of this continuum, the Coronavirus Disease-2019 (COVID-19) pandemic. The management of such cases must be carefully titrated, keeping in mind the risks and benefits of both pro-coagulant and anti-coagulant therapy wherein one can jeopardise the effects of the other.

Prospective Assessment of Biomarkers of Hypercoagulability in Oncological Patients and Healthcare Workers Following Vaccination Against Sars-Cov-2 with the mRNA Vaccine. the Roadmap-COVID-19-Vaccin Study

Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the ‘cytokine storm’ resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, p<0.001), D-Dimers (74% >UNL, p<0.01), vWF (60% >UNL, p<0.01), FVIII (62% >UNL, p<0.01) and shorter Procoag-PPL clotting time (96% <LNL, p<0.001), as compared to controls. Thrombin generation showed significantly higher Peak (60% >UNL, p<0.01), ETP (38% >UNL, p<0.01) and MRI (66% >UNL, p<0.01) but no differences in lag-time in vaccinated subjects as compared to the control group. Vaccinated subjects did not show any increase at the levels of TFa, TFPI, TM and E-selectin in comparison with the control group. The studied biomarkers were not significantly different between the VOnco and VHcw groups. Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even n patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures: Gligorov: Roche-Genentech: Research Funding;Novartis: Research Funding;Onxeo: Research Funding;Daichi: Research Funding;MSD: Research Funding;Eisai: Research Funding;Genomic Heatlh: Research Funding;Ipsen: Research Funding;Macrogenics: Research Funding;Pfizer: Research Funding. Terpos: Novartis: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Genesis: Consultancy, Honoraria, Research Funding;Celgene: Consultancy, Honoraria, Research Funding;BMS: Honoraria;Amgen: Consultancy, Honoraria, Research Funding;Takeda: Consultancy, Honoraria, Research Funding;Sanofi: Consultancy, Honoraria, Research Funding;GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria;BMS: Honoraria;Janssen: Honoraria;Beigene: Honoraria;Takeda: Honoraria.

SARS-COV2 [COVID-19]! A Cross Link between Coagulation and Inflammation

Introduction: In late 2019 a novel corona virus was identified thatreached pandemic proportions. The disease caused by the virus>Severe acute respiratory syndrome corona virus 2 [SARS-CoV-2]was designated as corona virus disease 2019 [COVID-19]. Covidpatients show excessive immune response [cytokine storm] as IL1,IL6, TNFa-chemokine levels are elevated. There is activation anddamage of the vascular endothelium, coagulation activation viamultiple pathways, dysregulation of natural anticoagulant pathway activation of white blood cells, platelets, Complement system.Aims &Objectives: As covid is associated with extreme inflammatory response,disordered haemostasis,high thrombotic risk our goalwas to start first line diagnostics investigations like haemostasis testssuch as PT, aPTT, Fibrinogen, D-dimer and pro inflammatory cytokine level such as IL 6 with CBC.Materials &Methods: Study done at tertiary hospital, Mumbai,India, 500 patients.M: F ratio: 1:1 [Age 18-96 yrs.], sample tested for PT, aPTT, Fibrinogen, D-Dimer, IL6 and Full blood count. Blood Collected:Sodium Citrate anticoagulant for PT, aPTT, Fibrinogen, D-Dimer.Tests analyzed quantitative on Stago Compact Max Instrument byusing Viscosity based detection system, latex enhanced immunoturbidimetric assay. IL-6 tested on Cobas E411 analyser usingElectrochemiluminescnce immunoassay. Full blood count: K2 EDTA,analysed on sysmex XN1000. WBC&Differential-Fluorescence Flowcytometry.Platelet count DC detection method, Immature plateletscount by flowresecent dye oxadine, Haemoglobin by SLS method.Inclusion criteria: patients with fever,cough,shortness of breath,bodyaches,sore throat,diarrhea,headache,Nausea or vomiting,loss of tasteand smell,congestion or runny nose. Exclusion Criteria: Samplegrossly haemolysed or received later than 4 h.Result:N: 50 Normal >10%] Covid test negative, all tested lab parametersnormal.N: 450 Positive [90%] Covid test positive.D-dimer [1000 ng/ml [markedly increased 3-fourfold over time].IL6 [25 pg/ml [markedly increased 3-fourfold over time].Neurophilia with low neutrophil lymphocyte ratio.Fibrinogen markedly elevated.Conclusions: D-Dimer increased in thrombotic events lead to fibrinolysis.Increased in IL-6 and Fibrinogen shows link betweeninflammation and procoagulant changes. In late stage thrombolysisdecreases fibrinogen levels and increase fibrin degradation products.If coagulation remains unchecked lead to consumptive coagulopathyand bleeding. Coagulation affects inflammatory activity and inflammation leads to coagulation activation so a cross link betweencoagulation and inflammation.

The Compass-COVID19-ICU Study: Identification of Factors to Predict the Risk of Intubation and Mortality in Patients with Severe COVID-19

Background: In some patients, SARS-CoV-2 infection induces cytokine storm, hypercoagulability and endothelial cell activation leading to worsening of COVID-19, intubation and death. Prompt identification of patients at risk of intubation or death is un unmet need. Objective: To derive a prognostic score for the risk of intubation or death in patients with critical COVID-19 by assessing biomarkers of hypercoagulability, endothelial cell activation and inflammation and a large panel of clinical analytes. Methods: We conducted a prospective, observational monocentric study enrolling 118 patients with COVID-19 admitted in the intensive care unit. At the 1st day of ICU admission all patients were assessed for the following biomarkers : protein C, protein S, antithrombin, D-Dimer, fibrin monomers, factors VIIa, V, XII, XII, VIII, von Willebrand antigen, fibrinogen, procoagulant phospholipid dependent clotting time, TFPI, thrombomodulin, P-selectin, heparinase, microparticles exposing tissue factor, IL-6, complement C3a, C5a, thrombin generation, prothrombin time, activated partial thromboplastin time, hemogram, platelet count) and clinical predictors. The clinical outcomes were intubation and mortality during hospitalization in ICU. Results: The intubation and mortality rate were 70 % and 18% respectively. Multivariate analysis led to the derivation of the COMPASS- COVID19-ICU score composed of P-Selectin, D-Dimer, free TFPI, TF activity, IL-6 and FXII, age and duration of hospitalization. The score predicted the risk of intubation or death with high sensitivity and specificity (0.90 and 0.92, respectively). Conclusions and Relevance: Critical COVID-19 is related with severe endothelial cell activation and hypercoagulability orchestrated in the context of inflammation. The COMPASS-COVID19-ICU score is an accurate predictive model for the evaluation of the risk of mechanical ventilation and death in patients with critical COVID-19. The assessment with the COMPASS- COVID-19-ICU score is feasible in tertiary hospitals. In this context it could be placed in the diagnostic procedure of personalized medical management and prompt therapeutic intervention. Disclosures: Terpos: Novartis: Honoraria;Janssen: Consultancy, Honoraria, Research Funding;Genesis: Consultancy, Honoraria, Research Funding;Celgene: Consultancy, Honoraria, Research Funding;BMS: Honoraria;Amgen: Consultancy, Honoraria, Research Funding;Takeda: Consultancy, Honoraria, Research Funding;Sanofi: Consultancy, Honoraria, Research Funding;GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria;BMS: Honoraria;Takeda: Honoraria;Beigene: Honoraria;Janssen: Honoraria.

Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation;however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4. Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained. Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14);or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14);or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results. Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors ith a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. [Formula presented] Disclosures: No relevant conflicts of interest to declare.