Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: 1. Application of Global Coagulation Assays and Platelet Function Testing in Dysfibrinogenemia and Hypodysfibrinogenemia and correlation with clinical phenotype. 2.SARS-CoV-2 Syndrome: application of coagulative parameters in Intensive Care Unit patients.

Abstract Project 1. Title. Application of Global Coagulation Assays and Platelet Function Testing in Dysfibrinogenemia and Hypodysfibrinogenemia and correlation with clinical phenotype Introduction. Dysfibrinogenemia (D) and hypodysfibrinogenemia (HD) are qualitative fibrinogen defects caused by heterozygosity for missense mutations in one of the three fibrinogen genes. Patients affected by D/HD present a wide variety of symptoms ranging from major bleeding to thrombotic complications. Asymptomatic patients are diagnosed because of a familial screening or laboratory pre-operatory/routine tests. Although there are some genotypes clearly associated to a higher thrombotic risk, the penetrance of the clinical phenotype is often incomplete suggesting there can be other genetic or environmental factors which can influence the clinical pattern. The available routine laboratory tests are useful to diagnose the disease but cannot predict the clinical phenotype: patients could develop, during life, hemorrhagic or thrombotic complications although they have been previously asymptomatic. Further information on the hemostatic potential of samples, in order to guide the therapy in this setting of patients, could be provided by the global coagulation assays, Thromboelastography (TEG) and Thrombin Generation Assay (TGA). In particular TEG measures the visco-elastic properties of a sample, during the whole hemostatic process. TGA gives information about thrombin production in a sample either in the initial or in the “burst” phase. A more accurate analysis of the hemostatic process, especially in the primary hemostasis, involves the contribution of platelets, whose fundamental and different functions can be investigated using Light Transmission Aggregometry (LTA). LTA measures the ability of platelets to aggregate, in response to different agonists. Aim. Aim of our study is to apply TEG and TGA to dysfibrinogenemia and hypodysfibrinogenemia samples from patients in baseline conditions, and to look for correlations between the clinical phenotype (hemorrhagic, thrombotic or asymptomatic), the genetic mutations and the laboratory parameters. In addition, the aim of the second part of the project is to apply LTA to the samples of patients with D/HD studied with global coagulation assays, to add information on primary hemostasis and its contribution to the D/HD clinical phenotype. Methods. The hemorrhagic phenotype is quantified using the ISTH-BAT score system [abnormal Bleeding Score (BS) > 3 in adult males and > 5 in adult females] and the EN-RBD-BSS. The thrombotic phenotype is also evaluated. Routine coagulations tests and Global Coagulation Assays, Thromboelastography (TEG®, Haemonetics) and Thrombin Generation Assay (CAT®, Stago), are applied to patient samples. TEG provided two protocols: Citrated Kaolin Reagent and Fibrinogen Reagent. In addition, Light Transmission Aggregometry is applied to patient samples in baseline conditions and the platelet agonists used were Adenosine Diphosphate (ADP, [5 μM]), Epinephrine (Ep, [10 μM]), Collagen (Coll, [2 μg/mL]), Arachidonic Acid (AA, [1 mM]). Results. To date, we have enrolled 10 patients (8 F, 2 M; median age 45 years, range 24 - 74). Routine coagulation tests confirmed the diagnosis of D / HD. According to medical investigation, 4 patients were considered asymptomatic and 6 with a bleeding diathesis. No one among the patients showed a significant ISTH-BAT-Bleeding Score (no hemorrhagic phenotype). The EN-RBD-BSS was suggestive of fibrinogen deficiency in only one patient while confirmed in all patients but one the diagnosis of D/HD. No patient had previous thrombosis. Eight patients were studied for the genetic causative mutation. TEG, Citrated Kaolin Reagent - Protocol: median R (n.v. 4.3 - 6.2 min) is increased (7 min, range 5.2 - 8.6); median K, α and MA are normal (K 1.9 min, range 1.08 - 4.6, n.v. 1.3 - 2.1; α 62.2°, range 40.1 - 64.7, n.v. 57.2 - 70.7 and MA 65.7 mm, range 48.7 - 69.7, n.v. 59.8 - 70.5 mm); Lysis parameters are decreased: median LY30 is 0.1% (range 0.0 - 0.1, n.v. 0.2 - 3.8) and median LY60 is 1.8% (range 0.0 - 2.7; n.v. 3.1 - 14.6%); Fibrinogen Reagent - Protocol: The R value is slightly increased: median R 3.2 min (range 2.2 - 4.8); also the MA parameter is slightly increased: median MA 21.1 min (range 4.5 - 28.0); K and α parameters are within the normal values: median K 10.1 min (range 3.3 - 17.3), median α 35.2° (range 11.0 - 53.3); Lysis results absent in all the patient analyzed: median LY30 is 0 % and median LY60 is 0 %. As regard TGA all the median parameters are normal: Lag Time 3.1 min (range 2.5 - 4.7, n.v. 1.9 - 3.9); ETP 1306.89 nM m (range 879.9 - 2011.4, n.v. 541.6 - 1484.1); Peak 218.4 nM (range 114.3 - 329.3, n.v. 90.1 - 258.5); tt-Peak 7.0 min (range 3.7 - 8.5, n.v. 3.8 - 8.7). Interestingly, ETP is increased in 4 patients and one of them presented heterozygosity for F II G 20210 A. No link between Global Coagulation Assays and Causative Mutations seemed to be found. Six patients of them have been studied also for LTA parameters. Median platelet count (n.v.: 150 - 450 x 103/μL) resulted normal (231 x 103/μL, range 188 - 257). All LTA median values were within normal ranges: median ADP = 65 (n.v.: 49 - 100%; range 44 - 91); median Ep = 67 (n.v.: 53 - 100%; range 36 - 89); median Coll = 78 (n.v.: 68 - 94%; range 19 - 84) and median AA = 91 (n.v.: 70 - 100%; range 4 - 98). patient had a severe reduction to 3 of the 4 agonists utilized (Ep = 36%, Coll = 19% and AA = 5%) while another patient presented a severe reduction only to AA (4%) without other alterations (ADP = 55%, Ep = 70% and Coll = 80%). No correlations with Clinical Phenotype nor Causative Mutations have been found. One patient had a severe reduction to 3 of the 4 agonists utilized (Ep = 36%, Coll = 19% and AA = 5%) while another patient presented a severe reduction only to AA (4%) without other alterations (ADP = 55%, Ep = 70% and Coll = 80%). No correlations with Clinical Phenotype nor Causative Mutations have been found. Conclusions. As regard TEG, the increased R is probably due to the type of qualitative mutations of the fibrinogen molecule and does not add other information than available routine tests. As regard TGA, the increased ETP in 4 patients seems not to be related to a thrombotic phenotype, but a prognostic value in situation at higher risk (major surgery, pregnancy, trauma, etc) in the same population cannot be excluded. Conversely, one of these 4 patients has been studied regarding platelet function: its LTA results were severely reduced to 3/4 agonists utilized, suggesting a bleeding phenotype. All the other patients manifested almost a normal LTA pattern. These preliminary results find a lack of correlation between both Global Coagulations Assays and LTA parameters with clinical phenotype as well as causative mutations. Longer follow-up and more enrolled patients are needed to confirm these preliminary data. Abstract Project 2. Title. SARS-CoV-2 Syndrome: application of coagulative parameters in Intensive Care Unit patients. Introduction. In December 2019, a novel coronavirus, later identified as SARS- CoV-2, caused a cluster of acute respiratory illnesses in Wuhan, Hubei Province, China. The pandemic scenario reported to April 2020 is that of a wide syndromic spectrum with different clinical manifestations: most patients had mild symptom and presented a good prognosis after the infection but, at the same time, up to 10 - 20% of cases (especially older people and those with underlying medical comorbidities) developed a severe disease (coronavirus disease 2019, COVID-19) characterized by interstitial pneumonia and the rapid development of acute respiratory distress syndrome (ARDS) or sepsis. The clinical picture of the severe SARS-CoV-2 patients was often associated with a hypercoagulability state with an increased risk of both venous and arterial thrombotic events and alteration of the hemostatic parameters: elevated D-dimer levels have been linked with a poor prognosis. The management of these thromboembolic complications is based on the use of heparin in the absence of contraindications (active bleeding and a platelet count of < 30 × 103 / μL). The efficacy, the dosage as well as the ratio between benefit and risk of the administration of heparin were controversial and remained to be defined. Aim. First aim of our study is to apply laboratory coagulative tests to samples from critical patients admitted to the Intensive Care Unit (ICU) of Policlinico Umberto I - Sapienza University Hospital in order to characterize the laboratory features of COVID-19 and evaluate the presence of a hypercoagulability state. To better understand the nature of this coagulopathy, especially with regard to endothelial involvement, von Willebrand factor parameters were evaluated. Secondary purpose is correlate these results and different doses of low-molecular-weight heparin (LMWH) administered to these patients as well as to register the rate of both venous and arterial thrombotic complications, in order to investigate the risk-benefit ratio of using heparin in severe COVID-19 patients. Patients. The patients were admitted to ICU with a severe SARS-CoV-2 infection (respiratory failure because of a bilateral interstitial pneumonia) without an active and diagnosed thromboembolic event. All patients were intubated and mechanically ventilated. Inclusion criteria were: age > 18 years, at least one positive swab for COVID-19 and assisted or controlled mechanical ventilation and PaO2/FiO2 < 200. Exclusion criteria: previous history of coagulopathy, antiplatelet therapy and important thrombocytopenia (platelet count < 50 × 103 / μL) or anemia (hemoglobin <10 g/dL). Methods. I cohort: two differently treated populations were observed: the “low- dose LMWH” (LD group), treated with 100 IU of LMWH/kg/day and the “high-dose LMWH” (HD group), treated with 100 IU of LMWH/kg/twice daily. The coagulative tests that were performed are PT, aPTT, Fibrinogen, AT, D-dimer, Platelet count, FVIII, vWF: Ag, vWF:RCo, Protein C and Protein S. Thrombin Generation Assay parameters were also evaluated. II cohort: patients were enrolled with no distinction of dose of LMWH. In this cohort of patients, we measured total levels of vWF:Ag and vWF:RCo. In addition, platelet aggregation in response to ADP, Collagen, Arachidonic Acid and platelet agglutination in response to ristocetin were evaluated and compared to ten healthy volunteers at laboratory of the Department of Experimental Medicine, Sapienza University of Rome. All the laboratory assays were evaluated on a single occasion. Results. I part: it was studied a consecutive series of 27 COVID-19 patients between April and May 2020. 17 males (63%) and 10 females (37%). Mean age: 66 years (range 38 - 85). LD group: 14 patients (51.9%); HD group: 13 patients (48.1%). Except the D-dimer, FVIII, vWF:Ag and vWF:RCo, all the median values of the coagulation tests were normal: PT ratio 1.12 (range 0.90 - 1.64); aPTT ratio 1.14 (range 0.81 - 3.06); Fibrinogen 383 mg/dL (range 62 - 663); AT 89 % (range 68 - 120); Platelet Count 214 x 103 / μL (range 51 - 378); PC 114 % (range 73 - 150); PS 71 % (range 36 - 98). The median value of D-dimer, FVIII, vWF:Ag and vWF:RCo resulted increased: 4686 ng/mL (range 465 - 35782), 209 % (range 109 - 393), 320 % (range 158 - 557); and 310 mg/dL (range 144 - 601 respectively. In the Thrombin Generation Assay, all the median values of the Lag Time and tt-Peak parameters resulted prolonged: Lag Time 7.7 min (range 3.0 - 32.2); tt-Peak 13.4 min (range 5.2 - 49.7). Conversely, all the median values of the endogenous thrombin potential (ETP) and Peak parameters resulted normal: ETP 953.5 nM x min (range 1.0 - 2357.2); Peak 122.2 nM (range 5.3 -268.5). Similar findings were reported between LD and HD patients, with no significative differences apart from ETP values (p < 0.01). Venous thromboembolism (VTE) rate was 11% (21% of LD patients - 0% in HD) while bleedings occurred in only one case (4%). II part: 19 patients, 10 (53%) males and 9 (47%) females. Mean age: 69 years (range 56 - 82). Both median vWF:Ag and vWF:RCo levels were higher than normal values: vWF:Ag 331 % (range 228 - 436) and vWF:RCo 322 mg/dL (range 172 - 471). As it regards platelet aggregation, the results showed that both ADP- and collagen-induced platelet aggregation were significantly reduced in COVID-19 patients, but not in response to arachidonic acid (0.5 mM) or ADP (4 μM) plus arachidonic acid (0.5 mM), used as maximal aggregation stimulus. An increase in the rates of agglutination induced by ristocetin (1.5 mg/mL) in patients COVID-19 patients was also reported. These results indicated a higher ability of vWF to bind to platelets. Conclusions. Our results confirmed the alteration of the hemostatic system in patients with severe SARS-CoV-2 infection: elevated D-dimer with/without thrombosis, absence of thrombocytopenia, elevated levels of FVIII and increased vWF tests (vWF:Ag and VWF:RCo) suggested the presence of a state of hypercoagulability associated with severe endothelial injury. In addition, lower response in platelet aggregation combined with a higher ability of vWF to bind to platelets might be able to lead to occlusion of microcirculation vessels. These coagulative parameters related to the proposed picture of Pulmonary Intravascular Coagulopathy (PIC) rather than Disseminate Intravascular Coagulation (DIC). Recently, the mechanism of the coagulopathy has been clarified and a peculiar pathophysiology entity has been proposed: COVID-19 associated Coagulopathy, CAC, characterized by hypercoagulability, platelet activation and endothelial dysfunction. Furthermore, we concluded that different doses of LMWH have an influence on the laboratory results, especially with regard to the total amount of thrombin generation, with a significant reduction in HD patients, decreasing the incidence of VTE without an increase in bleeding events. Randomized clinical trials were required to conclusively define the efficacy and safety of different doses of LMWH in patients with severe COVID-19 infection.