Intermediate dose enoxaparin in hospitalized patients with moderate-severe COVID-19: a pilot phase II single-arm study, INHIXACOVID19.

BACKGROUND: Randomized clinical trials in non-critically ill COVID-19 patients showed that therapeutic-dose heparin increased survival with reduced organ support as compared with usual-care thromboprophylaxis, albeit with increased bleeding risk. The purpose of the study is to assess the safety of intermediate dose enoxaparin in hospitalized patients with moderate to severe COVID-19. METHODS: A phase II single-arm interventional prospective study including patients receiving intermediate dose enoxaparin once daily according to body weight: 60 mg for 45-60 kg, 80 mg for 61-100 kg or 100 mg for > 100 kg for 14 days, with dose adjustment according to anti-factor Xa activity (target range: 0.4-0.6 UI/ml); an observational cohort (OC) included patients receiving enoxaparin 40 mg day for comparison. Follow-up was 90 days. Primary outcome was major bleeding within 30 and 90 days after treatment onset. Secondary outcome was the composite of all-cause 30 and 90-day mortality rates, disease severity at the end of treatment, intensive care unit (ICU) admission and length of ICU stay, length of hospitalization. All outcomes were adjudicated by an independent committee and analyzed before and after propensity score matching (PSm). RESULTS: Major bleeding was similar in IC (1/98 1.02%) and in the OC (none), with only one event observed in a patient receiving concomitantly anti-platelet therapy. The composite outcome was observed in 53/98 patients (54%) in the IC and 132/203 (65%) patients in the OC (p = 0.07) before PSm, while it was observed in 50/90 patients (55.6%) in the IC and in 56/90 patients (62.2%) in the OC after PSm (p = 0.45). Length of hospitalization was lower in the IC than in OC [median 13 (IQR 8-16) vs 14 (11-21) days, p = 0.001], however it lost statistical significance after PSm (p = 0.08). At 30 days, two patients had venous thrombosis and two pulmonary embolism in the OC. Time to first negative RT-PCR were similar in the two groups. CONCLUSIONS: Weight adjusted intermediate dose heparin with anti-FXa monitoring is safe with potential positive impact on clinical course in COVID-19 non-critically ill patients. TRIAL REGISTRATION: The study INHIXACOVID19 was registred on ClinicalTrials.gov with the trial registration number (TRN) NCT04427098 on 11/06/2020.

The M2 haplotype in the ANXA5 gene is an independent risk factor for idiopathic small-for-gestational age newborns.

Hereditary thrombophilias can impair vascular placental functions and predispose to the birth of small-for-gestational age (SGA) babies. The placental anticoagulant protein annexin A5 (ANXA5) may contribute to this process. A functional haplotype (M2) within the ANXA5 gene is associated with fetal loss and venous thrombosis. This study investigated the prevalence of the M2 haplotype in a group of women with idiopathic SGA newborn babies. Seventy-eight women with at least one previous unexplained SGA birth and 195 controls all from Southern Italy were investigated. Hereditary thrombophilia was found in 13 (16.5%) cases and 21 (11%) controls (P < 0.05.). The M2 haplotype was found in 29% of cases (n = 23) and 15% of controls [n = 30; P = 0.001; OR = 2.3, 95% CI (1.17-4.48)]. Within the case group, 82.5% of the M2 haplotype carriers gave birth to babies with a birthweight below the 3rd percentile [P = 0.01; OR = 2.4, 95% CI (1.26-4.73)]. A logistic regression, corrected for age, parity and gravity showed that the M2 haplotype was independently associated with the delivery of an SGA new born [P = 0.029; OR = 2.6, 95% CI (1.1-6.0)]. In conclusion, the M2 haplotype of the ANXA5 gene confers a risk of delivering SGA babies.

Thrombotic microangiopathy, DIC-syndrome and COVID-19: link with pregnancy prothrombotic state.

For last months, humanity has faced a formidable unknown enemy, which is presented as a new coronavirus infection. Despite the fact that the causative agents of new diseases appear at a certain frequency and that the virus SARS-CoV-2 has certain common properties with its predecessors, at the moment we are dealing with a new unknown pathogenesis of the development of severe complications in patients with risk factors. A final understanding of pathological process mechanisms is the goal of the scientific community. Summarizing research data from different countries, it became obvious that in severe cases of viral infection, we are dealing with a combination of the systemic inflammatory response syndrome, disseminated intravascular coagulation and thrombotic microangiopathy (TMA). Thrombotic microangiopathy is represented by a group of different conditions in which thrombocytopenia, hemolytic anemia, and multiple organ failure occur. The article reflects the main types of TMA, pathogenesis and principles of therapy. The main participants in the process are described in detail, including the von Willebrand factor and ADAMTS-13. Based on the knowledge available, as well as new data obtained from patients with COVID-19, we proposed possible models for the implementation of conditions such as sepsis, TMA, and DIC in patients with severe new coronavirus infection. Through a deeper understanding of pathogenesis, it will be possible to develop more effective diagnosis and therapy.

Infectious agents including COVID-19 and the involvement of blood coagulation and fibrinolysis. A narrative review.

OBJECTIVE: Platelets, blood coagulation along with fibrinolysis are greatly involved in the pathophysiology of infectious diseases induced by bacteria, parasites and virus. This phenomenon is not surprising since both the innate immunity and the hemostatic systems are two ancestral mechanisms which closely cooperate favoring host's defense against foreign invaders. However, the excessive response of these systems may be dangerous for the host itself. MATERIALS AND METHODS: We searched and retrieved the articles, using the following electronic database: MedLine and Embase. We limited our search to articles published in English, but no restrictions in terms of article type, publication year, and geography were adopted. RESULTS: The hemostatic phenotype of the infectious diseases is variable depending on the points of attack of the different involved pathogens. Infectious diseases which show a prothrombotic phenotype are bacterial sepsis, SARS-CoV-2 and malaria. However, among the bacterial sepsis, Yersinia Pestis is characterized by a profibrinolytic behavior. On the contrary, the hemorrhagic fevers, due to Dengue and Ebola virus, mainly exploit the activation of fibrinolysis secondary to a huge endothelial damage which can release a large amount of t-PA in the early phase of the diseases. CONCLUSIONS: Blood coagulation and fibrinolysis are greatly activated based on the strategy of the different infectious agents which exploit the excess of response of both systems to achieve the greatest possible virulence.

Effective and safe off-label use of caplacizumab treatment in a middle-aged obese male.

This study shows clinical efficacy and safety profile of an off-label use of caplacizumab for the treatment of immune-mediated thrombotic thrombocytopenic purpura in a middle-aged obese male patient manifesting aphasia, weakness and unconsciousness. Routine blood tests revealed haemolytic anaemia, severe thrombocytopenia (platelet count=20×109/L) and moderate creatinine increase. Diagnosis was based on the clinical judgement and laboratory determinations (undetectable ADAMTS13 activity and presence of anti-ADAMTS13 antibodies). The patient underwent plasma-exchange and an adjunctive treatment with prednisone (1mg/Kg/day), but the occurrence of a refractory and exacerbated form of disease suggested also using rituximab (375mg/m2 weekly for 4 weeks) and caplacizumab as salvage treatments. The caplacizumab was given at 10mg/day subcutaneously without the first intravenous bolus. Because von Willebrand factor inhibition, platelet count recovery and remission of symptoms were achieved, use of caplacizumab with this scheme appeared to be as effective as the approved one. Although this is an off-label use, this case highlights the potential of this new treatment, in terms of drug's efficacy and safety.

Factor XI deficiency: two novel mutations in asymptomatic Italian patients.

SUMMARY: Factor XI (FXI) deficiency is a rare bleeding disorder, resulting in a wide range of bleeding manifestations, from asymptomatic bleeding to injury-related bleeding. To identify mutations in FXI-deficient patients and to establish a possible relationship between clinical phenotype and genotype, we studied two patients from Southern Italy with FXI deficiency. They were identified by presurgical or routine laboratory screening. None of them showed bleeding. Three different mutations were detected (Glu117Stop, Cys118Arg and Trp497Gly); two of them were novel (Cys118Arg and Trp497Gly). One patient (with severe FXI levels) showed a compound heterozygosity (Glu117Stop with Cys118Arg). Two novel missense mutations were highly conserved among different species. In our patients, bleeding tendency did not appear to be correlated with FXI levels or with a single mutation in heterozygosis. On the other hand, the compound heterozygosis might explain low FXI levels, but it is not associated with bleeding. Our data confirm that a severe FXI deficiency is not necessarily associated with bleeding.

Venous thromboembolism during mycoplasma pneumoniae infection: case report and review of the literature.

Mycoplasma pneumoniae infection is frequent but generally mild or self-limiting. Approximately 10% of cases develop clinical signs of pneumonia with "atypical" radiographic pattern. However, mycoplasma pneumoniae can be responsible for a variety of extrapulmonary manifestations, potentially involving all systems and apparatuses. Although exact pathophysiological mechanisms are not completely known, these could be secondary to direct invasion of the target organ, immunological damage due to molecular mimicry or vascular obstruction. A 45-year-old man was admitted to Internal Medicine Unit because of fever, dry cough and fatigue lasting 15 days. Fever disappeared after starting clarithromycin. About 72 h after admission the patient complained of right calf pain and tachypnea. The presence of anti-mycoplasma antibodies suggested mycoplasma pneumoniae infection. Moreover, a diagnosis of venous thrombo-embolism was performed. Given the absence of classical risk factors for thrombosis, patient was investigated for inherited and acquired thrombophilia and tested positive for antiphospholipid antibodies. A review of the English literature on the association between m. pneumoniae and pulmonary embolism will be provided in order to underline the possible pathogenetic role of antiphospholipid antibodies in this setting. Clinicians should outweigh risk and benefits for LMWH prophylaxis case by case considering these adjunctive pro-thrombotic mechanisms in patients m. pneumoniae infection.

[Anticoagulant prophylaxis in women affected by thrombophilia and previous obstetric complications]. / Profilassi antitrombotica in donne con diagnosi di trombofilia e precedenti complicanze ostetriche.

Pregnancy is a condition of excessive clotting due to a decrease of some coagulation factors and a reduction of anticoagulant proteins, such as protein S. It is known that the causes of congenital or acquired thrombophilia may be associated with an increased risk of venous thromboembolism during pregnancy and/or obstetric complications, such early or late fetal loss, intrauterine fetal deaths, pre-eclampsia, fetal growth restriction. During pregnancy the use of a prophylaxis with antithrombotic drugs is considered at present a promising opportunity to significantly reduce the prevalence of thromboembolic complications, improving maternal and fetal outcomes. This article is a review to most recent evidence of pregnant anticoagulant prophylaxis in women with previous thromboembolic events.

Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization.

BACKGROUND: Standard methods of mutation detection are time consuming in Hemophilia A (HA) rendering their application unavailable in some analysis such as prenatal diagnosis. OBJECTIVES: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH) as an alternative and reliable tool for mutation detection in FVIII gene. PATIENTS/METHODS: We have applied a new method of cSBH that uses two different colors for detection of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously characterized individuals with FVIII deficiency. RESULTS: Data show that, when solution-phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be successfully detected. The technique was reliable also in identifying a mutant FVIII allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr) in exon 16 and three novel neutral polymorphisms are presented with an updated protocol for 2-color cSBH. CONCLUSIONS: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients.

Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet involvement and probucol-sensitive mechanism.

Homocystinuria due to homozygous cystathionine beta-synthase deficiency is an inborn error of metabolism characterized by a high incidence of thrombosis and premature atherosclerosis. We evaluated TXA2 biosynthesis in vivo and several in vitro tests of platelet function in 11 homocystinuric patients and 12 healthy controls. In vitro, patients' platelet aggregation was within control values as were TXB2 formation, fibrinogen binding, and ATP secretion in response to thrombin. In contrast, the urinary excretion of 11-dehydro-TXB2, a major enzymatic derivative of TXA2, was > 2 SD of controls in all patients (1,724 +/- 828 pg/mg creatinine, mean +/- SD, in patients vs. 345 +/- 136 in controls, P < 0.001). The administration to four patients of low-dose aspirin (50 mg/d for 1 wk) reduced metabolite excretion by > 80%. The recovery of 11-dehydro-TXB2 excretion over the 10 d that followed aspirin cessation occurred with a pattern consistent with the entry into the circulation of platelets with intact cyclooxygenase activity. Prolonged partial reduction in the abnormally high excretion of both 11-dehydro-TXB2 and 2,3-dinor-TXB2, was also observed in seven patients who ingested 500 mg daily for 3 wk of the antioxidant drug probucol. These results provide evidence for enhanced thromboxane biosynthesis in homocystinuria and for its partial dependence on probucol-sensitive mechanisms. Furthermore, the elevated TXA2 formation in homocystinuria is likely to reflect, at least in part, in vivo platelet activation.

The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis.

BACKGROUND: Myeloproliferative disorders (MPDs) represent a risk factor for thrombosis in the portal, mesenteric, and hepatic districts. OBJECTIVE: We aimed to assess whether the Janus kinase 2 (JAK2) V617F mutation, an acquired mutation that occurs in MPD patients, is a risk factor for portal and mesenteric venous thrombosis (PMVT) independently of the presence of overt MPDs. PATIENTS AND METHODS: The medical histories of 99 patients presenting with PMVT were obtained. The presence of the JAK2 V617F and VHL 598C > T mutations was determined by polymerase chain reaction followed by restriction enzyme analysis and direct cycle sequence analysis. RESULTS: Over a 10-year period of observation, of the 99 patients presenting with PMVT, the JAK2 V617F mutation was detected in heterozygous state in 17 individuals [17.2%; 95% confidence interval (95% CI) 10.9-25.9]. None of the patients presenting with the JAK2 V617F mutation carried an inherited thrombophilic risk factor. Seven patients with (43.8%; 95% CI 19.8-70.1) and two without (2.4%; 95% CI 0.3-8.4) the JAK2 V617F mutation had a diagnosis of MPD at the occurrence of the venous thrombotic event. After a median follow-up of 41 months (range 3-114 months), three out of the 10 patients carrying the JAK2 V617F mutation were then diagnosed as having idiopathic myelofibrosis (n = 2) or polycythemia vera (n = 1), whereas in seven patients a MPD was not detected. Two of the 83 patients without the JAK2 V617F mutation went on to develop MPDs. CONCLUSIONS: Determination of the JAK2 V617F mutation may contribute to the search for genetic determinants of PMVT and may be useful to recognize patients who should be carefully observed for the subsequent development of overt MPDs.

The G20210A prothrombin variant and the risk of venous thromboembolism or fetal loss in pregnant women: a family study.

BACKGROUND: The relationship between the G20210A prothrombin variant (PT-G20210A) and adverse pregnancy outcome has been studied by several groups in the last few years. However, because of the different design and sample sizes of these studies the estimated risks have varied. OBJECTIVE: In this retrospective, multi-center, cohort study we assessed the risk of thromboembolic or obstetric complications in women belonging to families of probands with isolated PT-G20210A and that were symptomatic for venous thromboembolism (VTE). METHODS: Two hundred and eighty-three female family members that had been pregnant at least once were enrolled. The occurrence of VTE and obstetric complications during pregnancy and postpartum were assessed in carriers of PT-G20210A and compared with non- carriers. RESULTS: One thromboembolic event occurred during the postpartum period in the carriers group. In the same group, 48 out of 359 pregnancies resulted in unexplained fetal loss as compared with 50 out of 357 pregnancies in the non-carriers (RR 0.9; 95% CI: 0.7-1.4). After adjustment, carriers of PT-G20210A showed a trend towards a higher risk of late fetal loss as compared with non-carriers (RR 2.2; 95% CI: 0.8-6.2). Furthermore, in pregnancies subsequent to those with previous fetal loss there was not a different risk of adverse outcome regardless of the carrier status. CONCLUSIONS: Female family members who are heterozygous carriers of isolated PT-G20210A do not seem to be at significant increased risk for fetal loss as compared with non-carriers. Screening for PT-G20210A of fertile age women belonging to these families is not warranted in this situation.

Low protein Z levels and risk of occurrence of deep vein thrombosis.

BACKGROUND: Protein Z (PZ) serves as a cofactor for activated factor X inhibition by the PZ-dependent protease inhibitor. In vivo and in vitro studies aimed at investigating the role of PZ levels in venous thombosis have produced conflicting results. OBJECTIVES: We investigated whether reduced PZ levels and PZ gene common variants are associated deep vein thrombosis (DVT). PATIENTS AND METHODS: In 197 patients with DVT and in 197 age-matched and sex-matched controls, PZ plasma levels and gene polymorphisms were evaluated by means of an enzyme-linked immunosorbent assay and direct cycle sequence analysis. RESULTS: Similar PZ levels were found in controls (1.44; SD 0.63 microg mL-1) and in patients (1.44; SD 0.96 microg mL-1). The incidence of PZ levels below the 5.0 (0.52 microg mL-1) or the 2.5 percentile of controls (0.47 microg mL-1) was higher in patients (10.2% and 8.7%, respectively) than in controls {4.1% [odds ratio (OR) 2.7, 95% confidence interval (CI) 1.2-7.3], and 2.0% (OR 4.6, 95% CI 1.5-13.9), respectively}. This relationship was independent of the effect of age, sex, and factor V Leiden and FII A(20210) alleles [OR 2.8 (95% CI 1.1-7.3), and OR 4.9 (95% CI 1.4-17.3)]. PZ levels were associated with the intron C G-42A and the intron F G79A polymorphisms in cases (r2=0.129) and in controls (r2=0.140). However, frequencies of the PZ gene polymorphisms were similar in the two groups and were not associated with very low PZ levels. CONCLUSIONS: The present data suggest an association between very low PZ plasma levels and the occurrence of DVT, with PZ gene polymorphisms contributing little to this relationship.

Genetic variations in the annexin A5 gene and the risk of pregnancy-related venous thrombosis.

BACKGROUND: Annexin A5 is a natural anticoagulant assumed to have thrombomodulary functions as it shields phospholipid layers from coagulation complexes. It was recently shown that the M2 haplotype within the annexin A5 gene (ANXA5) promoter reduces the transcriptional activity of the gene. In a previous report, the M2 haplotype was found to be associated with pregnancy-related venous thrombosis (VT). OBJECTIVES: To investigate whether the M1 or M2 haplotypes or other genetic variations in ANXA5 are associated with pregnancy-related VT. PATIENTS/METHODS: We investigated samples from 313 cases and 353 controls included in the VIP study, which is a case-control study of pregnancy-related VT. We analyzed tag single nucleotide polymorphisms (SNPs) selected from the CEU population (Utah Residents with Northern and Western European Ancestry) of HapMap and the M1 and the M2 haplotypes of the promoter. Odds ratios for VT were calculated for each haplotype with the wild type as the reference and for each tag SNP with the most common genotype as reference. RESULTS: We did not find any association between genetic variants in ANXA5 and the risk of pregnancy related VT, but some of the genetic variants were not in Hardy-Weinberg equilibrium. CONCLUSION: Neither the M1/M2 haplotypes nor the tag SNPs in ANXA5 were convincingly associated with pregnancy related VT, but other studies in this field are needed.

Clinical relevance of polymorphic markers of arterial thrombosis.

Case-control and cross-sectional studies show that some common molecular variations (polymorphisms) of genes coding for proteins involved in atherosclerosis and thrombosis are often present in subjects who have experienced cerebrovascular or cardiovascular events. The clinical impact of the majority of polymorphic markers is disputed by prospective reports. In contrast, their pathophysiological implications and their role in monitoring parameters that are difficult to be checked by alternative means, are documented by the large majority of the reports. From the evidence available, there may be suggestion for further impact of polymorphic markers in vascular medicine. To substantiate this, new prospective studies that include individuals from different geographical areas and that take into account the statistical power, the informativeness of the markers, the coexistance of established risk factors and the genetic background of the populations analyzed, are urgently needed.