Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory.

Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.

Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians.

Click to hear Dr Baglin's perspective on the role of the laboratory in treatment with new oral anticoagulants SUMMARY: One of the key benefits of the direct oral anticoagulants (DOACs) is that they do not require routine laboratory monitoring. Nevertheless, assessment of DOAC exposure and anticoagulant effects may become useful in various clinical scenarios. The five approved DOACs (apixaban, betrixaban, dabigatran etexilate, edoxaban and rivaroxaban) have different characteristics impacting assay selection and the interpretation of results. This article provides an updated overview on (i) which test to use (and their advantages and limitations), (ii) when to assay DOAC levels, (iii) how to interpret the results relating to bleeding risk, emergency situations and perioperative management, and (iv) what is the impact of DOACs on routine and specialized coagulation assays. Assays for anti-Xa or anti-IIa activity are the preferred methods when quantitative information is useful, although the situations in which to test for DOAC levels are still debated. Different reagent sensitivities and variabilities in laboratory calibrations impact assay results. International calibration standards for all specific tests for each DOAC are needed to reduce the inter-laboratory variability and allow inter-study comparisons. The impact of the DOACs on hemostasis testing may cause false-positive or false-negative results; however, these can be minimized by using specific assays and collecting blood samples at trough concentrations. Finally, prospective clinical trials are needed to validate the safety and efficacy of proposed laboratory thresholds in relation to clinical decisions. We offer recommendations on the tests to use for measuring DOACs and practical guidance on laboratory testing to help patient management and avoid diagnostic errors.

Inactivation of human coagulation factor X by a protease of the pathogen Capnocytophaga canimorsus.

Essentials Capnocytophaga canimorsus causes severe dog bite related blood stream infections. We investigated if C. canimorsus contributes to bleeding abnormalities during infection. The C. canimorsus protease CcDPP7 causes factor X dysfunction by N-terminal cleavage. CcDPP7 inhibits coagulation in vivo, which could promote immune evasion and trigger hemorrhage. SUMMARY: Background Capnocytophaga canimorsus is a Gram-negative bacterium that is present in the oral flora of dogs and causes fulminant sepsis in humans who have been bitten, licked, or scratched. In patients, bleeding abnormalities, such as petechiae, purpura fulminans, or disseminated intravascular coagulation (DIC), occur frequently. Objective To investigate whether C. canimorsus could actively contribute to these bleeding abnormalities. Methods Calibrated automated thrombogram and clotting time assays were performed to assess the anticoagulant activity of C. canimorsus 5 (Cc5), a strain isolated from a fatal human infection. Clotting factor activities were measured with factor-deficient plasma. Factor X cleavage was monitored with the radiolabeled zymogen and western blotting. Mutagenesis of Cc5 genes encoding putative serine proteases was performed to identify the protease that cleaves FX. Protein purification was performed with affinity chromatography. Edman degradation allowed the detection of N-terminal cleavage of FX. Tail bleeding times were measured in mice. Results We found that Cc5 inhibited thrombin generation and increased the prothrombin time and the activated partial thromboplastin time of human plasma via FX cleavage. A mutant that was unable to synthesize a type 7 dipeptidyl peptidase (DPP7) of the S46 serine protease family failed to proteolyse FX. The purified protease (CcDPP7) cleaved FX heavy and light chains from the N-terminus, and was active in vivo after intravenous injection. Conclusions This is, to our knowledge, the first study demonstrating a detailed mechanism for FX inactivation by a bacterial protease, and it is the first functional study associating DPP7 proteases with a potentially pathogenic outcome.

Facing coagulation disorders after acute trauma.

Facing coagulation disorders after acute trauma. PROBLEMS/OBJECTIVES: Trauma is the leading cause of mortality for persons between one and 44 years of age, essentially due to bleeding complications. METHODOLOGY: We screened the PubMed, Scopus and Cochrane Library databases, using specific keywords. Only publications in English were considered. MAIN RESULTS: The pathophysiology of trauma-induced coagulopathy (TIC) is complex and includes the classic "lethal triad" (i.e., haemodilution, acidosis, hypothermia) but may also include activation of protein C, endothelial and platelet dysfunction, and fibrinogen depletion. The time between trauma and treatment of the resultant massive bleeding should be as short as possible using techniques for rapid control of bleeding and avoiding aggravating factors (hypothermia, metabolic acidosis and hypocalcaemia). If given within three hours of injury, tranexamic acid (TXA) reduces all causes of mortality in trauma patients and reduces transfusion requirements. In a bleeding patient, crystalloids are preferred to colloids and the ratio of fresh frozen plasma to packed red blood cells should be at least 1:2. Damage control surgery (DCS) should be considered for patients who present with, or are at risk for developing, the "lethal triad", multiple life-threatening injuries or shock, and in mass casualty situations. DCS can also aid in the evaluation of the extent of tissue injuries and the control of haemorrhage and infection. Finally, there is currently no evidence of the added value of laboratory assays in the management of TIC. CONCLUSIONS: TIC appears quickly after trauma and should be anticipated and detected as soon as possible. TXA plays a central role in the management of such patients. Each institution should establish a local algorithm for the management of bleeding patients.

Recruitment and activation of circulating neutrophils after sinus surgery.

OBJECTIVE: After failure of pharmacological treatment, sinus surgery is the recommended alternative treatment for chronic sinusitis with or without nasal polyps. During post-operative healing, adequate local neutrophil activation plays an important role in the repair process. This pilot study aimed to systematically explore the participation of circulating neutrophils in early-phase wound repair of the nasal and paranasal mucosa after sinus surgery, with a special focus on neutrophil recruitment and activation patterns. METHODOLOGY: We conducted a single-center outcome study of patients undergoing sinus surgery. Whole blood samples were collected from eleven patients before surgery and at post-surgical time points of 1 hour and 1, 7, 14, and 30 days. Hematological analysis was conducted to count circulating neutrophils and evaluate their overall activation status. Using flow cytometry, neutrophil expression of membrane CD11b, CD11c, and CD15 was also measured, and oxidative burst analysis was performed. RESULTS: After sinus surgery, neutrophilia increased by 1 hour after surgery, reached a maximum at Day 1, and showed a gradual return toward baseline by Day 30. The oxidative burst initially decreased during the first hours after surgery, increased at Day 14, and returned toward normal by Day 30. Lewis X factor and the expression of CD11b and CD11c exhibited a bimodal change over time, in an inverted phase compared to the oxidative reaction. CONCLUSIONS: Circulating neutrophils are involved in the first phase of wound healing after sinus surgery as indicated by increased abundance, early membrane changes, and the modulation of their oxidative capacities.

Rapid exclusion of the diagnosis of immune HIT by AcuStar HIT and heparin-induced multiple electrode aggregometry.

BACKGROUND: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential but remains challenging. We have previously demonstrated, in a retrospective study, the usefulness of the combination of the 4Ts score, AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) with optimized thresholds. OBJECTIVES: We aimed at exploring prospectively the performances of our optimized diagnostic algorithm on suspected HIT patients. The secondary objective is to evaluate performances of AcuStar HIT-Ab (PF4-H) in comparison with the clinical outcome. METHODS: 116 inpatients with clinically suspected immune HIT were included. Our optimized diagnostic algorithm was applied to each patient. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) of the overall diagnostic strategy as well as AcuStar HIT-Ab (at manufacturer's thresholds and at our thresholds) were calculated using clinical diagnosis as the reference. RESULTS: Among 116 patients, 2 patients had clinically-diagnosed HIT. These 2 patients were positive on AcuStar HIT-Ab, AcuStar HIT-IgG and HIMEA. Using our optimized algorithm, all patients were correctly diagnosed. AcuStar HIT-Ab at our cut-off (>9.41 U/mL) and at manufacturer's cut-off (>1.00 U/mL) showed both a sensitivity of 100.0% and a specificity of 99.1% and 90.4%, respectively. CONCLUSION: The combination of the 4Ts score, the HemosIL® AcuStar HIT and HIMEA with optimized thresholds may be useful for the rapid and accurate exclusion of the diagnosis of immune HIT.

Platelet microparticle generation assay: a valuable test for immune heparin-induced thrombocytopenia diagnosis.

BACKGROUND: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance. OBJECTIVES: The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of (14)C-serotonin release assay (SRA) on the same series of patients. METHODS: Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1IU and 500IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient's medical records. Performances of PMPGA and SRA (n=53) were evaluated using ROC curve analysis with clinical outcome as reference. RESULTS: In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of (14)C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2). CONCLUSIONS: PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with (14)C-SRA performances and predominately with clinical outcome.

Assessment of the performances of AcuStar HIT and the combination with heparin-induced multiple electrode aggregometry: a retrospective study.

BACKGROUND: Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is challenging. HemosIL® AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) were recently proposed as rapid diagnostic methods. OBJECTIVES: We conducted a study to assess performances of AcuStar HIT-IgG (PF4-H) and AcuStar HIT-Ab (PF4-H). The secondary objective was to compare the performances of the combination of Acustar HIT and HIMEA with standardised clinical diagnosis. METHODS: Sera of 104 suspected HIT patients were retrospectively tested with AcuStar HIT. HIMEA was performed on available sera (n=81). The clinical diagnosis was established by analysing in a standardized manner the patient's medical records. These tests were also compared with PF4-Enhanced®, LTA, and SRA in subsets of patients. Thresholds were determined using ROC curve analysis with clinical outcome as reference. RESULTS: Using the recommended thresholds (1.00AU), the negative predictive value (NPV) of HIT-IgG and HIT-Ab were 100.0% (95% CI: 95.9%-100.0% and 95.7%-100.0%). The positive predictive value (PPV) were 64.3% (95% CI: 35.1%-87.2.2%) and 45.0% (95% CI: 23.2%-68.6%), respectively. Using our thresholds (HIT-IgG: 2.89AU, HIT-Ab: 9.41AU), NPV of HIT-IgG and HIT-Ab were 100.0% (95% CI: 96.0%-100.0% and 96.1%-100.0%). PPV were 75.0% (95% CI: 42.7%-94.5%) and 81.8% (95% CI: 48.3%-97.7%), respectively. Of the 79 patients with a medium-high pretest probability score, 67 were negative using HIT-IgG (PF4-H) test at our thresholds. HIMEA was performed on HIT-IgG positive patients. Using this combination, only one patient on 79 was incorrectly diagnosed. CONCLUSION: Acustar HIT showed good performances to exclude the diagnosis of HIT. Combination with HIMEA improves PPV.

Comparison of calibrated dilute thrombin time and aPTT tests with LC-MS/MS for the therapeutic monitoring of patients treated with dabigatran etexilate.

Ways to monitor dabigatran etexilate (DE) therapy would be useful in certain situations. Functional assays such as aPTT or Hemoclot® Thrombin Inhibitor (HTI) have been proposed to evaluate dabigatran concentrations, but previous findings are based on in vitro studies and results must be confirmed in clinical samples. The aim of this study was to compare aPTT and HTI measurements with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of dabigatran in plasma samples from DE treated patients. Seventy-one plasma samples were included. aPTT was performed using STA-CKPrest® and SynthASil®. HTI was performed according to instructions from the manufacturer. The LC-MS/MS method utilised dabigatran-d3 as internal standard. The plasma concentration range was 0 to 645 ng/ml as measured by LC-MS/MS. Overall, the HTI and LC-MS/MS analyses correlated well (r²=0.97). The Bland-Altman analysis showed a mean difference of 9 ng/ml (SD: 20 ng/ml). However, the HTI performed poorly at concentrations <50 ng/ml. LC-MS/MS was sensitive (limit of quantification 1.1 ng/ml) and specific for dabigatran. The aPTT methods did not correlate well with plasma concentrations measured by LC-MS/MS (r² = 0.59 with SynthASil® and 0.50 with STA-CKPrest®). In conclusion, the poor sensitivity, the important inter-individual variability, and the poor correlation with LC-MS/MS preclude the use of aPTT to estimate dabigatran concentrations. Due to its small inter-individual variability and good agreement with LC-MS/MS measurements, we recommend the use of HTI assays to rather accurately estimate concentrations of dabigatran >50 ng/ml. Quantification of lower dabigatran levels in DE-treated patients requires the "reference" LC-MS/MS method.

[Rivaroxaban: Xarelto--recommendations for pharmacists]. / Le Rivaroxaban (Xarelto): guide de la pratique officinale.

Rivaroxaban is one of the new oral anticoagulants (NOACs). It has many potential advantages in comparison with Vitamin K Antagonists (VKA). It has a predictable anticoagulant effect and does not theoretically require biological monitoring. It is also characterized by less food and drug interactions. However, due to major risks associated with over- and under-dosage, its optimal use in patients should be carefully followed by health care professionals. The aim of this article is to provide recommendations for pharmacists on the practical use of Xarelto in its different approved indications. This document is adapted from the practical user guide of rivaroxaban which was developed by an independent group of Belgian experts in the field of thrombosis and haemostasis.

Bioactivity and hemocompatibility study of amorphous hydrogenated carbon coatings produced by pulsed magnetron discharge.

Literature contains very few data about the potential biomedical application of amorphous hydrogenated carbon (a-C:H) thin films deposited by reactive pulsed magnetron discharge even so it is one of the most scalable plasma deposition technique. In this article, we show that such a C2H2 pulsed magnetron plasma produces high quality coating with good hemocompatibility and bioactive response: no effect on hemolysis and hemostasis were observed, and proliferation of various cell types such as endothelial, fibroblast, and osteoblast-like cells was not affected when the deposition conditions were varied. Cell growth on a-C:H coatings is proposed to take place by a two-step process: the initial cell contact is affected by the smooth topography of the a-C:H coatings, whereas the polymeric-like structure, together with a moderate hydrophilicity and a high hydrogen content, directs the posterior cell spreading while preserving the hemocompatible behavior.

[Clinical studies, the interests and limits of using dabigatran in atrial fibrillation]. / Dabigatran dans la fibrillation auriculaire: etudes cliniques, intérêts et limites.

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia, especially in older people. This condition is associated with an increased risk of stroke, and long-term anticoagulation treatment is therefore needed. Vitamin K antagonists are effective in reducing the risk of stroke but optimal use of these drugs remains difficult. The development of new oral anticoagulant drugs is therefore highly relevant. Dabigatran is an oral direct thrombin inhibitor. Its prodrug, dabigatran etexilate, is marketed under the name of Pradaxa and was initially approved for the prevention of thromboembolic events in major orthopedic surgery. It has been recently approved for stroke prevention in patients with AF. The purpose of this paper is to review--in light of current knowledge--the interests and limits of using dabigatran etexilate in AF. Briefly, dabigatran etexilate is not inferior to warfarin in AF. However many questions remain unanswered, including questions related to the concomitant use of dabigatran etexilate and acetylsalicylic acid, the possible increased risk of myocardial infarction and the need for drug monitoring.

BM-520, an original TXA2 modulator, inhibits the action of thromboxane A2 and 8-iso-prostaglandin F2alphain vitro and in vivo on human and rodent platelets, and aortic vascular smooth muscles from rodents.

Thromboxane A(2) (TXA(2)) and 8-iso-PGF(2alpha) are two prostanoid agonists of the thromboxane A(2) receptor (TP), whose activation has been involved in platelet aggregation and atherosclerosis. Agents able to counteract the actions of these agonists are of great interest in the treatment and prevention of cardiovascular events. Here, we investigated in vitro and in vivo the pharmacological profile of BM-520, a new TP antagonist. In our experiments, this compound showed a great binding affinity for human washed platelets TP receptors, and prevented human platelet activation and aggregation induced by U-46619, arachidonic acid and 8-iso-PGF(2alpha). The TP receptor antagonist property of BM-520 was confirmed by its relaxing effect on rat aorta smooth muscle preparations precontracted with U-46619 and 8-iso-PGF(2alpha). Further, its TP antagonism was also demonstrated in vivo in guinea pig after a single intravenous injection (10 mg kg(-1)). We conclude that this novel TP antagonist could be a promising therapeutic tool in pathologies such as atherosclerosis where an increased production of TXA(2) and 8-iso-PGF(2alpha), as well as TP activation are well-established pathogenic events.

Confirmation of high cytokine clearance by hemofiltration with a cellulose triacetate membrane with large pores: an in vivo study.

OBJECTIVE: To confirm in vivo the hypothesis that hemofiltration with a large pore membrane can achieve significant cytokine clearance. METHOD: We used a well-known animal model of endotoxinic shock (0.5 mg/kg of lipopolysaccharide from Escherichia Coli over a period of 30 mins). Six pigs were hemofiltrated for 3 hours with a large pore membrane (78 A pore, 80 kDa cut off) (Sureflux FH 70, Nipro, Osaka, Japan). The ultrafiltration rate was 45 ml/kg/min. Samples were taken from arterial, venous line and in the ultrafiltrate at T120 and T240. We measured concentrations of interleukin 6, interleukin 10 and albumin. RESULTS: At T120 and T240, the IL-6 clearances were 22 +/- 7 and 15 +/- 3 ml/min, respectively. The IL-6 sieving coefficients were 0.97 and 0.7 at T120 and T240, respectively. At T120 and T240, the IL-10 clearances were 14 +/- 4 and 10 +/- 7 ml/min, respectively. The sieving coefficients were 0.63 and 0.45 at T120 and T240, respectively. The concentrations of IL-6 and IL-10 were the same at T0 and T240. At T60 and T240, the plasmatic albumin concentrations were 24 +/- 4 g/L and 23 +/- 4 g/L, respectively (p = 0.13). CONCLUSIONS: In this animal model of endotoxinic shock, we confirm the high cytokine clearance observed when hemofiltration is applied to a large pore membrane. The loss of albumin seems negligible. The impact of such clearances on hemodynamic stability and survival remains to be proved.

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors.

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.