Pharmacodynamics Monitoring of Emicizumab in Patients with Hemophilia A.

BACKGROUND: Emicizumab is a bispecific antibody mimicking coagulation factor VIII (FVIII) employed to treat patients with hemophilia A (PwHA) regardless of FVIII inhibitor status. The identification of biological markers reflecting the hemostatic competence of patients under emicizumab therapy would have a great clinical value. Unfortunately, emicizumab over-corrects standard coagulation assays, precluding their use for evaluating the hemostatic correction achieved in vivo. Here, we investigated whether global coagulation assays (GCA) would allow monitoring the biological response to non-factor replacement therapy with emicizumab. MATERIALS AND METHODS: Six adults PwHA received a weekly dose of emicizumab of 3 mg/kg during weeks (W) 1 4 and 1.5 mg/kg from W5 onwards. Response to treatment was monitored weekly by emicizumab plasma concentration, thrombin generation (TG), and fibrin clot formation (FCF) and structure. TG and FCF results were compared to patient baseline, FVIII replacement, and healthy donors. RESULTS: TG and FCF significantly increased in PwHA after the loading period, reaching a plateau that lasted until the end of monitoring. Similarly, fibrin clot network became denser with thinner fibrin fibers. However, TG contrary to FCF remained at the lower limits of reference values. Remarkably, despite having similar plateau concentrations of emicizumab some patients showed markedly different degrees of TG and FCF improvement. CONCLUSION: Our study enriches the knowledge on the use of GCA to monitor non-factor replacement therapy, indicating that TG and FCF could act as direct markers of emicizumab biological activity. GCA allow to capture and visualize the individually variable response to emicizumab, leading a step forward to the personalization of patient treatment.

Thrombocytopathies: Not Just Aggregation Defects-The Clinical Relevance of Procoagulant Platelets.

Platelets are active key players in haemostasis. Qualitative platelet dysfunctions result in thrombocytopathies variously characterized by defects of their adhesive and procoagulant activation endpoints. In this review, we summarize the traditional platelet defects in adhesion, secretion, and aggregation. In addition, we review the current knowledge about procoagulant platelets, focusing on their role in bleeding or thrombotic pathologies and their pharmaceutical modulation. Procoagulant activity is an important feature of platelet activation, which should be specifically evaluated during the investigation of a suspected thrombocytopathy.

High-Dose Epinephrine Enhances Platelet Aggregation at the Expense of Procoagulant Activity.

Platelet activation is characterized by shape change, granule secretion, activation of fibrinogen receptor (glycoprotein IIb/IIIa) sustaining platelet aggregation, and externalization of negatively charged aminophospholipids contributing to platelet procoagulant activity. Epinephrine (EPI) alone is a weak platelet activator. However, it is able to potentiate platelet activation initiated by other agonists. In this work, we investigated the role of EPI in the generation of procoagulant platelets. Human platelets were activated with convulxin (CVX), thrombin (THR) or protease-activated receptor (PAR) agonists, EPI, and combination thereof. Platelet aggregation was assessed by light transmission aggregometry or with PAC-1 binding by flow cytometry. Procoagulant collagen-and-THR (COAT) platelets, induced by combined activation with CVX-and-THR, were visualized by flow cytometry as Annexin-V-positive and PAC-1-negative platelets. Cytosolic calcium fluxes were monitored by flow cytometry using Fluo-3 indicator. EPI increased platelet aggregation induced by all agonist combinations tested. On the other hand, EPI dose-dependently reduced the formation of procoagulant COAT platelets generated by combined CVX-and-THR activation. We observed a decreased Annexin-V-positivity and increased binding of PAC-1 with the triple activation (CVX + THR + EPI) compared with CVX + THR. Calcium mobilization with triple activation was decreased with the higher EPI dose (1,000 µM) compared with CVX + THR calcium kinetics. In conclusion, when platelets are activated with CVX-and-THR, the addition of increasing concentrations of EPI (triple stimulation) modulates platelet response reducing cytosolic calcium mobilization, decreasing procoagulant activity, and enhancing platelet aggregation.

Heparin-Induced Thrombocytopenia: A Review of New Concepts in Pathogenesis, Diagnosis, and Management.

Knowledge on heparin-induced thrombocytopenia keeps increasing. Recent progress on diagnosis and management as well as several discoveries concerning its pathogenesis have been made. However, many aspects of heparin-induced thrombocytopenia remain partly unknown, and exact application of these new insights still need to be addressed. This article reviews the main new concepts in pathogenesis, diagnosis, and management of heparin-induced thrombocytopenia.

Tissue Factor-Independent Coagulation Correlates with Clinical Phenotype in Factor XI Deficiency and Replacement Therapy.

BACKGROUND: In factor XI (FXI) deficiency, bleeding cannot be predicted by routine analyses. Since FXI is involved in tissue factor (TF)-independent propagation loop of coagulation, we hypothesized that investigating the spatiotemporal separated phases of coagulation (TF-dependent and -independent) could improve diagnostics. OBJECTIVES: This article investigates the correlation of parameters describing TF-dependent and -independent coagulation with the clinical phenotype of FXI deficiency and their ability to assess hemostasis after FXI replacement. METHODS: We analyzed: (1) plasma from healthy controls (n = 53); (2) normal plasma (n = 4) spiked with increasing concentrations of a specific FXI inhibitor (C7P); (3) plasma from FXI-deficient patients (n = 24) with different clinical phenotypes (13 bleeders, 8 non-bleeders, 3 prothrombotics); (4) FXI-deficient plasma spiked with FXI concentrate (n = 6); and (5) plasma from FXI-deficient patients after FXI replacement (n = 7). Thrombin generation was measured with the reference method calibrated automated thrombogram and with Thrombodynamics (TD), a novel global assay differentiating TF-dependent and -independent coagulation. RESULTS: C7P dose-dependently decreased FXI activity, prolonged activated partial thromboplastin time, and hampered TF-independent coagulation. In FXI-deficient bleeders, TD parameters describing TF-independent propagation of coagulation and fibrin clot formation were reduced compared with controls and FXI-deficient nonbleeders and increased in FXI-deficient patients with prothrombotic phenotype. Receiver operating characteristic analysis indicated that TF-independent parameters were useful for discriminating FXI-deficient bleeders from non-bleeders. In FXI-deficient plasma spiked with FXI concentrate and in patients receiving FXI replacement, TD parameters were shifted toward hypercoagulation already at plasma FXI levels around 20%. CONCLUSION: TF-independent coagulation parameters assessed by TD have the potential to identify the clinical phenotype in FXI-deficient patients and to monitor FXI replacement therapy.

Sodium-Calcium Exchanger Reverse Mode Sustains Dichotomous Ion Fluxes Required for Procoagulant COAT Platelet Formation.

Procoagulant collagen-and-thrombin (COAT)-activated platelets represent a subpopulation of activated platelets, which retain a coat of prohemostatic proteins and express phosphatidylserine on their surface. Dichotomous intracellular signaling generating procoagulant platelet activity instead of traditional aggregating endpoints is still not fully elucidated. It has been demonstrated that secondary messengers such as calcium and sodium play a critical role in platelet activation. Therefore, we developed a flow cytometric analysis to investigate intracellular ion fluxes simultaneously during generation of aggregating and procoagulant platelets. Human platelets were activated by convulxin-plus-thrombin. Cytosolic calcium, sodium, and potassium ion fluxes were visualized by specific ion probes and analyzed by flow cytometry. We observed high and prolonged intracellular calcium concentration, transient sodium increase, and fast potassium efflux in COAT platelets, whereas aggregating non-COAT platelets rapidly decreased their calcium content, maintaining higher cytosolic sodium, and experiencing lower and slower potassium depletion. Considering these antithetical patterns, we investigated the role of the sodium-calcium exchanger (NCX) during convulxin-plus-thrombin activation. NCX inhibitors, CBDMB and ORM-10103, dose-dependently reduced the global calcium mobilization induced by convulxin-plus-thrombin activation and dose-dependently prevented formation of procoagulant COAT platelets. Our data demonstrate that both NCX modes are used after convulxin-plus-thrombin-induced platelet activation. Non-COAT platelets use forward-mode NCX, thus pumping calcium out and moving sodium in, while COAT platelets rely on reverse NCX function, which pumps additional calcium into the cytosol, by extruding sodium. In conclusion, we described for the first time the critical and dichotomous role of NCX function during convulxin-plus-thrombin-induced platelet activation.

Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia.

Patients affected by the rare Glanzmann thrombasthenia (GT) suffer from defective or low levels of the platelet-associated glycoprotein (GP) IIb/IIIa, which acts as a fibrinogen receptor, and have therefore an impaired ability to aggregate platelets. Because the procoagulant activity is a dichotomous facet of platelet activation, diverging from the aggregation endpoint, we were interested in characterizing the ability to generate procoagulant platelets in GT patients. Therefore, we investigated, by flow cytometry analysis, platelet functions in three GT patients as well as their ability to generate procoagulant collagen-and-thrombin (COAT) platelets upon combined activation with convulxin-plus-thrombin. In addition, we further characterized intracellular ion fluxes during the procoagulant response, using specific probes to monitor by flow cytometry kinetics of cytosolic calcium, sodium, and potassium ion fluxes. GT patients generated higher percentages of procoagulant COAT platelets compared to healthy donors. Moreover, they were able to mobilize higher levels of cytosolic calcium following convulxin-plus-thrombin activation, which is congruent with the greater procoagulant activity. Further investigations will dissect the role of GPIIb/IIIa outside-in signalling possibly implicated in the regulation of platelet procoagulant activity.

Predicting Venous Thromboembolic Events in Patients with Coronavirus Disease 2019 Requiring Hospitalization: an Observational Retrospective Study by the COVIDIC Initiative in a Swiss University Hospital.

BACKGROUND: Coronavirus disease 2019 (COVID-19) can result in profound changes in blood coagulation. The aim of the study was to determine the incidence and predictors of venous thromboembolic events (VTE) among patients with COVID-19 requiring hospital admission. Subjects and Methods. We performed a retrospective study at the Lausanne University Hospital with patients admitted because of COVID-19 from February 28 to April 30, 2020. RESULTS: Among 443 patients with COVID-19, VTE was diagnosed in 41 patients (9.3%; 27 pulmonary embolisms, 12 deep vein thrombosis, one pulmonary embolism and deep vein thrombosis, one portal vein thrombosis). VTE was diagnosed already upon admission in 14 (34.1%) patients and 27 (65.9%) during hospital stay (18 in ICU and nine in wards outside the ICU). Multivariate analysis revealed D-dimer value > 3,120 ng/ml (P < 0.001; OR 15.8, 95% CI 4.7-52.9) and duration of 8 days or more from COVID-19 symptoms onset to presentation (P 0.020; OR 4.8, 95% CI 1.3-18.3) to be independently associated with VTE upon admission. D-dimer value ≥ 3,000 ng/l combined with a Wells score for PE ≥ 2 was highly specific (sensitivity 57.1%, specificity 91.6%) in detecting VTE upon admission. Development of VTE during hospitalization was independently associated with D-dimer value > 5,611 ng/ml (P < 0.001; OR 6.3, 95% CI 2.4-16.2) and mechanical ventilation (P < 0.001; OR 5.9, 95% CI 2.3-15.1). CONCLUSIONS: VTE seems to be a common COVID-19 complication upon admission and during hospitalization, especially in ICU. The combination of Wells ≥ 2 score and D - dimer ≥ 3,000 ng/l is a good predictor of VTE at admission.

How to Capture the Bleeding Phenotype in FXI-Deficient Patients.

Factor XI (FXI) is a serine protease involved in the propagation phase of coagulation and in providing clot stability. Several mutations in the F11 gene lead to FXI deficiency, a rare mild bleeding disorder. Current laboratory methods are unable to assess bleeding risk in FXI-deficient patients, because the degree of bleeding tendency does not correlate with plasma FXI activity as measured by routine coagulometric aPTT-based assays. Bleeding manifestations are highly variable among FXI-deficient patients and FXI replacement therapy can be associated with an increased thrombotic risk. A correct evaluation of the patient hemostatic potential is crucial to prevent under- or overtreatment. In recent years, different research groups have investigated the use of global coagulation assays as alternative for studying the role of FXI in hemostasis and identifying the clinical phenotype of FXI deficiency. This brief review article summarizes the main features of coagulation factor XI and its deficiency and resumes the principle axes of research and methods used to investigate FXI functions.

Biomarkers of liver dysfunction correlate with a prothrombotic and not with a prohaemorrhagic profile in patients with cirrhosis.

BACKGROUND & AIMS: Different liver dysfunction biomarkers are used to assess the bleeding risk of patients with cirrhosis, either as such or included in bleeding risk assessment scores. Since the current model of coagulation in patients with cirrhosis describes a procoagulant tendency with increasing severity according to Child-Pugh stage, we decided to investigate the relation between liver dysfunction biomarkers and thrombin generation. Our aim was to verify their adequacy for bleeding risk assessment. METHODS: We performed a prospective single-centre study including 260 patients with liver cirrhosis. Thrombin generation was measured using ST Genesia® Thrombin Generation System without and with thrombomodulin in order to assess the role of proteins C and S. Relations between thrombin generation and Child-Pugh/model for end-stage liver disease (MELD) scores, prothrombin time (PT)/international normalised ratio (INR), activated partial thromboplastin time (aPTT), factor V activity, albumin, and total bilirubin were assessed. RESULTS: Thrombomodulin-mediated inhibition of thrombin generation was significantly decreased in patients with liver cirrhosis compared with healthy donors (p <0.0001) and in Child-Pugh B and C compared with A (p <0.0001 [A-B], 0.4515 [B-C], <0.0001 [A-C]). Thrombomodulin-mediated inhibition significantly decreased with increasing PT/INR, aPTT, and total bilirubin levels and with decreasing factor V activity and albumin levels. CONCLUSIONS: Worsening liver dysfunction biomarkers reflect an increasing prothrombotic profile in patients with liver cirrhosis. In particular, prolonged PT/INR and aPTT as well as decreasing factor V activity are related to an increasing thrombotic risk and not to an increasing bleeding risk. These parameters should not be used to assess bleeding risk due to haemostatic anomalies in patients with liver cirrhosis. Alternative biomarkers for bleeding risk assessment in patients with liver cirrhosis need to be developed. LAY SUMMARY: We demonstrate that the laboratory parameters used to assess bleeding risk of patients with liver disease, e.g. prothrombin time/international normalised ratio (PT/INR) and activated partial thromboplastin time (aPTT), are inadequate for this purpose because they are correlated with a prothrombotic coagulation profile. In this article, we highlight the need for alternative parameters to assess bleeding risk in patients with liver disease.

[Hematology in the time of COVID-19]. / L'hématologie au temps du COVID-19.

The COVID-19 pandemic impacts the hematology practice. Intensive chemotherapies for high-grade lymphomas and acute leukemias, multiple myeloma treatments and most hematopoietic stem cell transplantations should be performed as usual. Low-grade lymphomas should only be treated when strictly indicated, maintenance can be postponed. Other myeloid neoplasia and their therapies cause imunosupression; dose adjustment is recommended but no brisk stopping. Sickle cell anemia patients are highly succeptible to severe COVID-19 course. Thrombocytopenia and procoagulant state are associated with severe courses of COVID-19, requiring an individualized therapy. No data indicate a risk of SARS-CoV-2 transmission through blood product transfusion.

La pandémie de COVID-19 affecte la prise en charge hématologique. Les chimiothérapies intensives pour les lymphomes agressifs et les leucémies aiguës, les traitements du myélome multiple, ainsi que la plupart des greffes de cellules souches hématopoïétiques doivent continuer à être pratiquées. Les lymphomes de bas grade seront traités uniquement avec des indications claires ; et la maintenance repoussée. Les autres néoplasies myéloïdes et leurs traitements causent une immunosuppression ; on recommande une adaptation des doses, mais pas d'arrêt brusque. La drépanocytose rend les patients très vulnérables au COVID-19. La thrombopénie signe un état procoagulant et la sévérité du COVID-19, nécessitant un traitement individualisé. Aucune donnée n'indique de risque d'une transmission du SARS-CoV-2 par transfusion de produits sanguins.

Thrombin generation in a woman with heterozygous factor V Leiden and combined oral contraceptives: A case report.

Combined oral contraceptives and factor V Leiden mutation are multiplicative risk factors for venous thromboembolism. However, it remains unknown whether this multiplicative effect is reflected in thrombin generation assays. We report here the evolution of the thrombin generation profile while taking combined oral contraceptives and after their discontinuation in a woman with heterozygous factor V Leiden mutation. The proband exhibited a distinctly prothrombotic thrombin generation profile including markedly decreased thrombomodulin (TM) sensitivity, compared to the control population. This profile possibly reflected a high thrombotic risk. After discontinuation of combined oral contraceptives, thrombin generation and TM sensitivity improved greatly, leaving only a slightly prothrombotic profile. Therefore, the multiplied thrombotic risk occurring with simultaneous combined oral contraceptives and factor V Leiden mutation is reflected by a thrombin generation assay performed without and with TM. This could be a promising tool to identify women taking combined oral contraceptives at high risk for venous thromboembolism. Further studies are needed to verify this hypothesis.

Hemostatic Alterations in Patients With Cirrhosis: From Primary Hemostasis to Fibrinolysis.

In the setting of liver cirrhosis (LC), profound hemostatic changes occur, which affect primary hemostasis, coagulation, and fibrinolysis. They involve prohemorrhagic and prothrombotic alterations at each of these steps. Patients with cirrhosis exhibit multifactorial thrombocytopenia and in vitro thrombocytopathy, counterbalanced by increased von Willebrand factor. The resultant shift is difficult to assess, but overall these changes probably result in a rebalanced primary hemostasis. Concerning coagulation, the reduced activity of coagulation factors is counterbalanced by an increase in factor VIII (produced by liver sinusoidal endothelial cells), a decrease of the natural anticoagulants, and complex changes, including changes in circulating microparticles, cell-free DNA, and neutrophil extracellular traps. Overall, these alterations result in a procoagulant state. As for fibrinolysis, increased tissue-type and urokinase-type plasminogen activators, a relatively decreased plasminogen activator inhibitor 1, and decreased levels of thrombin-activatable fibrinolysis inhibitor and α2-antiplasmin are counterbalanced by decreased plasminogen and a decreased fibrin clot permeability. Whether and how these changes shift fibrinolysis remains to be determined. Overall, the current consensus is that in patients with cirrhosis, the hemostasis is shifted toward a procoagulant state. We review the published evidence for the concept of LC as a prothrombotic state, discuss discordant data, and highlight the impact of the underlying cause of LC on the resultant imbalance.

Rapid and accurate Bayesian diagnosis of heparin-induced thrombocytopenia.

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT.

Hyper-coagulability in obese patients accurately identified by combinations of global coagulation assay parameters.

BACKGROUND: Routine laboratory methods are insensitive to hyper-coagulation, which may be detected by global hemostasis tests. Calibrated Automated Thrombogram (CAT) is a gold standard method to measure thrombin generation and coagulation potential. Thrombodynamics (TD) is a new global assay that monitors the spatio-temporal propagation of blood coagulation, separating initiation from amplification/propagation phases of coagulation and visualizing fibrin clot formation. AIM: We investigated whether CAT and/or TD can identify hyper- and hypo-coagulable states in patients with well-characterized phenotypes and which parameters could be used as potential predictors of thrombotic risk. METHODS: Blood was collected from: (1) forty healthy volunteers; (2) twelve obese patients scheduled for bariatric surgery (BMI ≥ 35 kg/m2); (3) nine patients under therapy with vitamin K-antagonists (median INR 2.7); (4) eight patients treated with low molecular weight heparins (anti-Xa activity between 0.5 and 0.7 U anti-Xa/ml); (5) ten patients with hemophilia A or B. Tissue factor induced thrombin generation was measured with CAT. Propagation of thrombin generation and clot growth from a tissue factor coated surface were monitored with TD. RESULTS: Thrombin generation and fibrin clot formation parameters were significantly higher in obese patients compared to healthy volunteers and anticoagulated or hemophilic patients. ROC analysis of combined CAT or CAT/TD parameters (integrating thrombin generation and fibrin clot formation) demonstrated an excellent accuracy in detecting hyper-coagulability. CONCLUSION: Combinations of CAT assay parameters and of parameters of thrombin generation burst with final fibrin clot properties allow recognizing accurately hyper-coagulable plasma and may represent predictive markers for thrombotic events.

Episodes of fever in neutropenia in pediatric patients with cancer in Bern, Switzerland, 1993-2012.

Fever in neutropenia (FN) is the most frequent potentially life threatening complication of chemotherapy for cancer. Prediction of the risk to develop complications, integrated into clinical decision rules, would allow for risk-stratified treatment of FN. This retrospective, single center cohort study in pediatric patients diagnosed with cancer before 17 years, covered two decades, 1993 to 2012. In total, 703 FN episodes in 291 patients with chemotherapy (maximum per patient, 9) were reported here. Twenty-nine characteristics of FN were collected: 6 were patient- and cancer-related, 8 were characteristics of history, 8 of clinical examination, and 7 laboratory results in peripheral blood, all known at FN diagnosis. In total 28 FN outcomes were assessed: 8 described treatment of FN, 6 described microbiologically defined infections (MDI), 4 clinically defined infections, 4 were additional clinical composite outcomes, and 6 outcomes were related to discharge. These data can mainly be used to study FN characteristics and their association with outcomes over time and between centers, and for derivation and external validation of clinical decision rules.

Pediatric patients at risk for fever in chemotherapy-induced neutropenia in Bern, Switzerland, 1993-2012.

Fever in neutropenia (FN) is the most frequent potentially life threatening complication of chemotherapy for cancer. Prediction of the risk to develop FN during chemotherapy would allow for targeted prophylaxis. This retrospective, single centre cohort study in pediatric patients diagnosed with cancer before 17 years covered two decades, 1993 to 2012. The 583 (73%) of 800 patients diagnosed with cancer who had received chemotherapy were studied here. Data on 2113 observation periods was collected, defined by stable combinations of 11 predefined characteristics potentially associated with FN. They covered 692 years of cumulative chemotherapy exposure time, during which 712 FN episodes were diagnosed, 154 (22%) of them with bacteremia. The risk to develop FN and FN with bacteremia remained stable over time. These data can mainly be used to study FN risks over time and between centers, and to derive or externally validate FN risk prediction rules.