Blood Coagulation & Fibrinolysis

Cellular immune dysregulation in the pathogenesis of immune thrombocytopenia Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by immune-mediated increased platelet destruction and decreased platelet production, resulting from immune intolerance to autoantigen. The pathogenesis of ITP remains unclear, although dysfunction of T and B lymphocytes has been shown to be involved in the pathogenesis of ITP. More recently, it is found that dendritic cells, natural killer, and myeloid-derived suppressor cells also play an important role in ITP. Elucidating its pathogenesis is expected to provide novel channels for the targeted therapy of ITP. This article will review the role of different immune cells in ITP.

A novel missense mutation in F9 gene causes hemophilia B in a family with clinical variability Hemophilia B is an X-linked recessive bleeding disorder caused by diverse mutations throughout the F9 gene. The same F9 mutation may result in different degrees of clotting factor deficiency. The aim of this study was to investigate the pathogenesis of two hemophilia B patients with different severity in a family. A family with two hemophilia B patients was recruited in this study. Coagulation assays, activities of FVIII (FVIII:C) and FIX (FIX:C) were evaluated. All of the exons and intron exon boundaries of the F9 gene were amplified by PCR and analyzed by direct sequencing. The proband, 12-year-old boy with moderate bleeding history, had manifest prolonged activated partial thromboplastin time (98.1 s) and markedly decreased FIX activity (1%). His maternal uncle presented slightly prolonged activated partial thromboplastin time (48.2 s) and mildly decreased FIX activity (15.2%). Molecular genetic analysis of F9 revealed that they were hemizygous for a novel missense mutation, c.157G>C (p.Glu53Gln). Our study widens the mutation spectrum of the FIX gene. In addition, this report provides a specific case associated with genotype and phenotype heterogeneity of hemophilia B.

Thrombin generation and thromboelastometry in monitoring the in-vitro reversal of warfarin: a comparison between 3-factor and 4-factor prothrombin complex concentrates The efficacy of three-factor prothrombin complex concentrates (PCCs) in the reversal of vitamin K antagonists is still a matter of debate. We compared the ‘in-vitro’ effect of three PCCs (one three-factor and two four-factor) on international normalized ratio (INR), thrombin generation and thromboelastometry of patients at different degrees of anticoagulation with vitamin K antagonist. We tested three concentrations of PCC (0.5, 1 and 1.5 U/ml) in six patients: three (INR 2.0–2.9) and three (INR 3.0–4.0). In this preliminary phase, we determined the lowest effective dose for a target INR less than 1.5 and to normalize endogenous thrombin potential and clotting time in EXTEM assay. In the validation phase, we tested the effect of the newly determined lowest effective PCC dose on samples of 40 (INR 2.0–2.9) and 20 (INR 3.0–4.0) patients. The minimum efficacious dosage to achieve the target INR with three-factor PCC (3-PCC) was 0.5 (INR 2.0–2.9) and 1.5 U/ml (INR 3.0–4.0). Four-factor PCCs (4-PCCs) achieved target INR with the lowest dose (0.5 U/ml) independently of baseline INR. Thrombin generation endogenous thrombin potential and EXTEM clotting time achieved normal values with the lowest dose (0.5 U/ml) of either 3-PCC or 4-PCC independently of baseline INR. Data observed in the preliminary phase were confirmed in the validation phase. 3-PCC appears to be as effective as 4-PCC in reversing oral anticoagulant treatment based on thrombin generation and EXTEM data, but not INR data, at least in the range of INR considered in our study. Further studies are needed to address the clinical implications of our results.

The effect of mycophenolate mofetil on platelet function Mycophenolate mofetil (MMF) raises platelet counts in patients with primary immune thrombocytopenia. However, studies indicate that MMF inhibits collagen-induced platelet aggregation, potentially increasing bleeding risk following MMF therapy. The study evaluates the in-vitro effect of MMF on platelet function. Blood samples (n = 6) from healthy donors were incubated with vehicle, MMF or mycophenolic acid (MPA) at clinically relevant concentrations. Platelet aggregation was measured with flow cytometry and 96-well light transmission aggregometry (LTA). Using flow cytometry, we measured the expression of platelet CD49b, CD42b, CD42a, CD61 and CD41. Platelet activation was measured as the expression of P-selectin and the active form of the GPIIb/IIIa receptor following agonist stimulation. Agonists were: ADP, thrombin receptor-activating peptide, collagen, collagen-related peptide and U46619. The Platelet Function Analyzer-200 was used to measure global platelet function. MMF and MPA did not change platelet aggregation regardless of the agonist used. An exception was a significant, but minor decrease in collagen-induced platelet aggregation in samples with MMF (6 ± 3%, P = 0.02) and MPA (8 ± 4%, P = 0.01) compared with vehicle (22 ± 11%). However, this was not observed using the lesser sensitive LTA method. Compared with vehicle, MPA led to a significantly lower relative disposition of the surface collagen-receptor GPVI (7.8 ± 1.8 versus 8.8 ± 2.1 mean fluorescence intensity, P < 0.001). In all other platelet-related tests, neither MMF nor MPA showed any effect. In conclusion, MMF and MPA only had a minor effect on collagen-induced platelet aggregation, with MPA reducing the relative disposition of surface GPVI receptors.

Comparison of international normalized ratio determined by point-of-care to standard laboratory testing before and after reversal of heparin in cardiac surgery Study Objective To compare point-of-care (POC) of international normalized ratio to laboratory-derived values before and after cardiopulmonary bypass, with the primary aim of evaluating for any change in the relationship between the tests. Methods This is a prospective observational study with 50 patients undergoing cardiac surgery enrolled. The International normalized ratio measured at two time points, precardiopulmonary bypass and after heparin reversal with protamine using both POC i-STAT and standard laboratory analysis for both time points. A difference of 0.2 between tests at either time point was considered clinically significant based on previous literature. A paired t test was used to test for a changing or statistically significant mean difference between tests. At both time points values were categorized into absolute difference of more than 0.2 or less than 0.2, and a Fisher's exact test was used to determine if an association existed between heparin reversal and a difference more than 0.2. Bland–Altman plots were also evaluated for agreement. Results A statistically and clinically significant mean difference [0.09 vs. 0.25, difference −0.163 95% confidence interval (−0.25, −0.08), P = 0.003] was seen between the laboratory and POC tests when pre and postheparin reversal samples were compared. A significantly greater number of patients had a clinically relevant difference between the tests post compared with pre (four patients vs. 18 patients, P = 0.001). Linear regression analysis of the difference compared with the means, showed significant correlation suggesting the presence of a proportional bias (pre r = 0.488, P = <0.01, post r = 0.571, P = <0.01). Conclusion Clinically significant differences exist between POC and laboratory testing of international normalized ratio after heparin reversal during cardiac surgery. ClinicalTrials.gov Identifier NCT03267823.

A comparison of coagulation test results from heparinized central venous catheter and venipuncture Blood sampling via heparin-locked central venous catheter, including coagulation tests, is possible in accordance with the Clinical & Laboratory Standards Institute guidelines. However, differences exist between the test values of samples obtained from central venous catheter and those obtained from peripheral veins, even the guidelines are followed. To compare the coagulation time between blood samples from the heparin-locked central venous catheter and peripheral veins. In total, 72 hospitalized patients using heparin-locked Hickman catheters were enrolled. Blood samples for coagulation testing were simultaneously obtained via the peripheral veins and heparin-locked Hickman catheters. For sampling from the catheters, 0.9% sodium chloride flushing was performed and 10 or 23 ml of blood was discarded prior to collecting the coagulation test samples. Correlation, Bland–Altman plot, covariate, and regression analysis were performed for data analyses. Despite following the guidelines, the activated partial thromboplastin time test values differed. In the 10 ml of blood discard group, a correlation coefficient of 0.378 and a mean bias of 6.46 s were determined, while and in the 23 ml blood discard group, a correlation coefficient of 0.80 and a mean bias of 2.518 s were determined. Therefore, the volume of blood discarded from the heparin-locked Hickman catheters may affect the activated partial thromboplastin time test values.

Plasma levels of enoxaparin oligosaccharides, antifactor-Xa and thrombin generation in patients undergoing haemodialysis Low molecular weight heparins are used during haemodialysis for thromboprophylaxis of the dialysis circuit, with plasma antifactor-Xa (anti-Xa) activity used as a surrogate measure for effective anticoagulation. However, this pharmacokinetic parameter does not always correlate with pharmacodynamic effects in patients. The aim of this study was to investigate the relationship between actual plasma levels of the low molecular weight heparins enoxaparin, anti-Xa activity, and global coagulation measurement of thrombin generation during haemodialysis. Blood was analysed from 16 adult patients with end-stage kidney disease at 0, 2, 4 h, and at completion of 31 dialysis sessions where single fixed doses of 20 (n = 3), 40 (n = 16), 60 (n = 6), or 80 (n = 6) mg of enoxaparin (equating to 0.23–1.07 mg/kg) were used as thromboprophylaxis. Plasma enoxaparin oligosaccharides [degree of polymerization (dp)6–dp16] were measured by high-performance size exclusion chromatography, anti-Xa activity by colourimetric assay, and thrombin generation by calibrated automated thrombogram. Plasma enoxaparin fragments were undetectable at the beginning of each dialysis, peaked at 2 h to levels that correlated with dose (r = 0.68, P < 0.001) then remained relatively stable. In contrast, therapeutic anti-Xa levels achieved at 2 h in 18 cases (58%) quickly dropped to only six cases (19%) at the end of dialysis, by which time thrombin generation had also recovered in 81% of patients. Statistical modelling revealed a threshold value of anti-Xa at 0.53 IU/ml that supressed thrombin generation to 15.28% of baseline (P < 0.001). Despite loss of anticoagulant activity in the majority of patients, plasma levels of enoxaparin oligosaccharides remained detectable and relatively unchanged throughout dialysis.

Gene variants in four pedigrees with hereditary coagulation factor XI deficiency and one novel mutation identification Coagulation factor XI (FXI) deficiency is a bleeding disorder with unpredictable severity. Patients with this condition usually suffer bleeding manifestations after trauma or surgery and are poorly correlated with plasma FXI activity (FXI:C). In the current study, we examined and identified the phenotype and genotype in four unrelated probands and their 32 relatives with hereditary FXI deficiency. The probands with severely reduced FXI:C but bleeding symptoms were only found in two probands. Mutation analysis showed that all the probands were FXI homozygous mutation or compound heterozygous mutation. Five mutations were identified including three nonsense mutations c.841C>T (p.Gln263X), c.1107C>A (p.Tyr351X) and c.1033A>T (p.Lys327X), respectively, one frameshift mutation c.1325delT (p.Leu424CysfsX8), and one splicing mutation c.326-1G>A. c.1033A>T (p. Lys327X), a novel mutation which lead to a premature stop codon at amino acid position 327, it may have an influence on protein characteristics and cause the corresponding disease.

Heparin challenge test in patients undergoing cardiac surgery: dealing with heparin allergy A history of heparin hypersensitivity in patients undergoing cardiopulmonary bypass surgery poses the dilemma of which anticoagulant to use. Here, we report the successful use of a heparin challenge test in a 66-year-old female candidate for coronary artery bypass graft surgery with a past medical history of enoxaparin type I hypersensitivity after pulmonary embolism. Challenge and desensitization protocols are effectively used for essential antibiotics in patients with severe infections and/or allergies, or patients with aspirin intolerance requiring revascularization for coronary disease. A successful use of desensitization protocols to unfractionated heparin has been previously described in four patients undergoing cardiac surgery with various schemes. However, our case report indicates that a challenge test may also offer a quick, safe and effective approach in patients with a history of hypersensitivity reactions to heparin with inconclusive diagnostic tests and/or whenever the use of alternative heparins is tricky.

Thromboelastography-targeted management of severe coagulopathy and off-label use of four-factor prothrombin complex concentrate in an infant with massive bleeding A 4-month-old girl initially presented to the pediatric ICU at 9 days old with multiorgan failure and cerebellar hemorrhage secondary to disseminated enteroviral infection and was eventually listed for liver transplant. Due to severe coagulopathy, she developed a hemothorax after line placement. Despite operative exploration and multiple recombinant factor VIIa doses, massive bleeding continued. The bleeding was finally controlled with thromboelastography-targeted platelet and cryoprecipitate transfusion in addition to four-factor prothrombin complex concentrate administration. This management strategy was invaluable in controlling bleeding from an iatrogenic cause.