Genetic Background of von Willebrand Disease: History, Current State, and Future Perspectives.

Sequencing of the gene encoding for von Willebrand factor (VWF) has brought new insight into the physiology of VWF as well as its pathophysiology in the context of von Willebrand disease (VWD). Molecular testing in VWD patients has shown high variability in the overall genetic background of this condition. Almost 600 mutations and many disease-causing mechanisms have been described in the 35 years since the VWF gene was identified. Genetic testing in VWD patients is now available in many centers as a part of the VWD diagnostic algorithm. Molecular mechanisms leading to types 2 and 3 VWD are well characterized; thus, information from genetic analysis in these VWD types may be beneficial for their correct classification. However, the molecular basis of type 1 VWD is still not fully elucidated and most likely represents a multifactorial disorder reflecting a combined impact of environmental and genetic factors within and outside of VWF. Regarding sequencing methods, the previous gold-standard Sanger sequencing is gradually being replaced with next-generation sequencing methods that are more cost- and time-effective. Instead of gene-by-gene approaches, gene panels of genes for coagulation factors and related proteins have recently become a center of attention in patients with inherited bleeding disorders, especially because a high proportion of VWD patients, mainly those with low VWF plasma levels (type 1), appear to be free of mutations in VWF. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are accessible in a very limited number of laboratories. Results from these studies have presented several genes other than VWF or ABO possibly affecting VWF levels, and such findings will need further validation studies.

Effect of Bisoprolol on the Level of Dabigatran.

BACKGROUND: Many patients receiving dabigatran treatment might also require bisoprolol therapy. However, there is a possibility that bisoprolol as significant P-glycoprotein inhibitor might interact with dabigatran. STUDY QUESTION: To investigate the impact of concomitant bisoprolol therapy on dabigatran plasma level in patients with nonvalvular atrial fibrillation. STUDY DESIGN: A pilot drug interaction study in 29 patients with nonvalvular atrial fibrillation on dabigatran therapy. Bisoprolol was administrated in 18 patients. Blood samples were collected at baseline (in the morning, before any medication was administered) and at hour 2 (2 hours after administration of dabigatran and bisoprolol). RESULTS: The dabigatran plasma level was significantly higher at baseline and at hour 2 in patients treated with bisoprolol compared with patients without bisoprolol therapy. In addition, we have shown that this increase is affected by dabigatran dosage and concomitant treatment with proton-pump inhibitor and digoxin. The impact of bisoprolol on dabigatran concentration was still significant despite these confounders. CONCLUSIONS: This study demonstrated the interaction between dabigatran and bisoprolol, which is modulated with dabigatran dosage and concomitant treatment with proton-pump inhibitor and digoxin.

Edoxaban affects TRAP-dependent platelet aggregation.

Edoxaban is an oral anticoagulant drug and a direct factor Xa inhibitor. However, it is still not fully understood if and how edoxaban impacts platelet function. This prospective study aimed to assess in vitro platelet function in patients with atrial fibrillation (AF) receiving edoxaban. It was a single centre study quantifying platelet aggregation in 20 patients treated with edoxaban by light transmission aggregometry. The thrombin receptor activating peptide (TRAP)-induced platelet aggregation was significantly lower 2 h after taking edoxaban compared to baseline value (44.7 ± 32.03% vs. 73.3 ± 25.55%; p < 0.0001). In addition, we did not find any significant difference in results between the patient groups.The TRAP-induced platelet aggregation is reduced in non-valvular AF patients receiving edoxaban.

The Impact of Proton Pump Inhibition on Dabigatran Levels in Patients With Atrial Fibrillation.

BACKGROUND: Proton pump inhibition (PPI) administrated together with dabigatran reduces the risk of gastrointestinal hemorrhage. However, there is a discussion regarding possible PPI-dabigatran interaction that may reduce the efficacy of this therapy. STUDY QUESTION: To determine the impact of concomitant PPI on dabigatran plasma levels in patients with nonvalvular atrial fibrillation (NV-AF). STUDY DESIGN: A pilot prospective study in patients with NV-AF on dabigatran therapy was performed; 31 patients were enrolled. PPI with either omeprazole or pantoprazole was administrated in 19 patients. MEASURES AND OUTCOMES: Blood samples were taken for the assessment of the dabigatran trough and peak levels. Dabigatran concentration was measured with the Hemoclot Thrombin Inhibitor Assay. RESULTS: There were significant differences in dabigatran trough level comparing patients treated with PPI and patients without PPI (58.86 ± 36.76 ng/mL vs. 110.72 ± 88.47 ng/mL, P < 0.05). Similarly, there were significant differences in dabigatran peak level between compared groups (88.0 ± 20.5 ng/mL vs. 174.4 ± 139.64 ng/mL, P < 0.05). CONCLUSIONS: This pilot study demonstrated the interaction between PPI and dabigatran levels in patients with NV-AF.

First evidence: rivaroxaban and apixaban reduce thrombin-dependent platelet aggregation.

Rivaroxaban and apixaban are direct oral anticoagulants whose target specificity is to activate factor X (FXa). It is still not fully understood how xabans impact platelet function. This single-center observational study aimed to assess in vitro platelet function in patients with atrial fibrillation receiving rivaroxaban or apixaban. It examined quantification of platelet aggregation assessed by light transmission aggregometry in thirty-four patients treated with apixaban or rivaroxaban. The thrombin-induced platelet aggregation was significantly lower 2 h after taking selected xabans compared to baseline value (69.55 ± 32.15% vs. 44.79 ± 34.97.9%; p < 0.0001). This effect was only observed in patients who received rivaroxaban or apixaban for more than 1 week. The thrombin-induced platelet aggregation is reduced in cardiovascular patients receiving rivaroxaban or apixaban. This reduction is likely to depend on the duration of the treatment. Duration of treatment should be considered in future studies focusing on DOACs and platelet aggregation.

Dabigatran affects thrombin-dependent platelet aggregation after a week-long therapy.

OBJECTIVE: Dabigatran is a direct thrombin inhibitor. As the main adverse event is bleeding, it is relevant whether dabigatran has additional effects on platelet function. If so, it could affect the bleeding risk. We aimed to assess in vitro aggregation in patients with atrial fibrillation (AF) receiving dabigatran. DESIGN: We evaluated 32 AF patients treated with dabigatran (study group) and 18 non-anticoagulated non-AF blood donors (control group). We assessed light transmittance platelet aggregation (LTA) with 100 nmol/L γ-thrombin in both groups. The LTA was performed at two time-points in our dabigatran group of patients. RESULTS: The thrombin-induced platelet aggregation was significantly lower two hours after dabigatran was taken compared to baseline measurement (9% ± 6% vs. 29% ± 21%) in our study group. Moreover, we observed that the baseline value of platelet aggregation in patients on dabigatran treatment was significantly lower compared to healthy volunteers (29% ± 21% vs. 89 ± 8). However, one subanalysis showed that this significant reduction in platelet aggregation at baseline was only observed in patients who received dabigatran for over a week. CONCLUSION: The thrombin-induced platelet aggregation is reduced in non-valvular AF patients receiving dabigatran after a week-long therapy.

Heparin-induced Thrombocytopenia Presenting With Deep Venous Thrombosis and Pulmonary Embolism Successfully Treated With Rivaroxaban: Clinical Case Report and Review of Current Experiences.

Heparin-induced thrombocytopenia (HIT) is a life or limb-threatening thrombotic thrombocytopenia. HIT is traditionally treated with factor-IIa inhibitors such as bivalirudin, lepirudin, or argatroban. However, these agents usually require parenteral administration and are not generally available in all countries. Recently, several experiences with novel oral anticoagulants (NOACs) administration to treat HIT had been reported. NOACs generally offer advantages such as consistent and predictable anticoagulation, oral administration with good patient compliance, and a good safety profile. We report a case of HIT with severe thrombotic complications successfully treated with rivaroxaban and discuss the current knowledge about the use of NOACs for the treatment of this potentially fatal thrombocytopenia.

Monitoring of dabigatran therapy using Hemoclot(®) Thrombin Inhibitor assay in patients with atrial fibrillation.

Dabigatran, a new direct thrombin inhibitor, achieves strong anticoagulation that is more predictable than warfarin. Nevertheless, a patient on dabigatran therapy (DT) may suffer from thrombotic or bleeding events. The routine monitoring of DT is not recommended, and standard coagulation tests are not sensitive enough for the assessment of DT activity. The aim of this study was to examine the clinical usefulness of the Hemoclot(®) Thrombin Inhibitor (HTI) assay in the assessment of dabigatran plasma levels in patients with non-valvular AF. Nineteen patients (12 men, 7 women) on DT were included in this preliminary prospective observational study. Dabigatran was administrated twice daily in a two dose regimens: 150 mg (5 patients) and 110 mg (14 patients). Blood samples were taken for the assessment of trough and peak levels of dabigatran. Dabigatran concentrations were measured with the HTI assay. The average dabigatran trough level was 69.3 ± 55.5 ng/ml and the average dabigatran peak level was 112.7 ± 66.6 ng/ml. The dabigatran trough plasma concentration was in the established reference range in 15 patients and the dabigatran peak plasma concentration was in the established reference range in 9 patients, respectively. Despite the fact that the activated partial thromboplastin and thrombin times were generally changed (prolonged), these tests failed to identify the patients with too low or too high dabigatran concentrations. The study confirmed the high sensitivity of the HTI assay for the assessment of dabigatran plasma levels. When compared to standard coagulation tests, the HTI is a more suitable assay for the monitoring of patients treated with dabigatran. Monitoring of DT may be beneficial in selected patients; however, further studies will be needed for the final clarification of this issue.

How can Secondary Thromboprophylaxis in High-Risk Pregnant Patients be Improved?

Low-molecular-weight heparin (LMWH) is suggested for thromboprophylaxis in pregnant women with previous venous thromboembolism (VTE). Anyway, there is only limited amount of studies evaluating the effect of LMWH on hemostatic parameters during pregnancy of patients with previous VTE and the need of secondary thromboprophylaxis. We therefore provide results of prospective and longitudinal assessment of changes in hemostasis in high-risk pregnant women at four times during pregnancy (T1-T4) and one time after the postpartum period (T5) used for individualized modification of thromboprophylaxis. In this study, the results of coagulation factor VIII (FVIII) and protein S (PS) activity, ProC Global ratio and anti-Xa activity were evaluated. Despite the thromboprophylaxis, an increased predisposition to thromboembolic complications was detected (significant increase in FVIII activity and decrease in PS function, ProC Global ratio not normalized even after the postpartum period - p < .0001 between controls and T5 for PS and ProC Global). These results indicate that hemostasis may not be restored even 6 to 8 weeks after delivery and pose the question when is it safe to withdraw the anticoagulant thromboprophylaxis in high-risk patients with prior VTE.

Impact of Dabigatran Treatment on Rotation Thromboelastometry.

A rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. The aim of this study was to study the anticoagulant effects of dabigatran in patients with atrial fibrillation (AF) as assessed by the whole blood assays ROTEM, and how data from these methods correlate to plasma dabigatran concentrations measured by Hemoclot. ROTEM was performed with ROTEM Gamma (Pentapharm GmbH, Munich, Germany). The assays used in our study were Ex-tem and In-tem assay. Plasma dabigatran concentrations were determined by hemoclot thrombin inhibitor assay (Hyphen BioMed, France) at trough and post-dose in 27 patients on dabigatran 150 mg BID. Median plasma dabigatran concentrations at trough were 74 ng/mL (11.2-250) and post-dose (2 h after ingestion) 120 ng/mL (31-282). The ROTEM clotting time (CT) and maximum clot firmnes (MCF) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (p < 0.0001) and In-tem (p < 0.0001). In summary, in our study, we have found that the ROTEM variable CT and MCF, when activated with triggers Ex-tem and In-tem, has a strong and highly significant correlation with the plasma dabigatran concentration in a real-life population of AF-patients and could thereby be an alternative to estimate dabigatran concentration in emergency situations. However, additional studies are needed to further validate these findings.

How Can Rotational Thromboelastometry as a Point-of-Care Method Be Useful for the Management of Secondary Thromboprophylaxis in High-Risk Pregnant Patients?

Thromboprophylaxis with low-molecular-weight heparin (LMWH) for patients with a history of venous thromboembolism (VTE) is suggested. Rotational thromboelastometry (ROTEM®) represents an innovative point-of-care method enabling the complex and quick evaluation of hemostasis. However, there are only episodic cases of its use for hemostasis assessment and guidance of LMWH in pregnancy. Therefore, we provide the results of unique prospective and longitudinal monitoring of hemostasis in high-risk pregnant women, which we used for the individualized optimalization of secondary thromboprophylaxis. According to the shortening of clot formation time (CFT) in EXTEM (p = 0.0007 from the 26th gestational week vs. controls) and INTEM (p = 0.002 from the 35th gestational week), increase in alpha angle (AA) in EXTEM, INTEM, and HEPTEM, and the persistence of increase in maximum clot firmness (MCF) in EXTEM, INTEM, and HEPTEM (p < 0.001 from the 26th and 35th gestational week vs. controls for EXTEM and INTEM, p = 0.0012 from the 26th gestational week in HEPTEM), LMWH dose was modified. Even after the postpartum period, AA in EXTEM was steeper than in controls (p = 0.0007), indicating that hemostasis is not fully normalized after 6-8 weeks following delivery. Therefore, ROTEM may be a useful tool for the individual evaluation of the termination of anticoagulant thromboprophylaxis.

Impact of Edoxaban on Thrombin-Dependent Platelet Aggregation.

Edoxaban, a direct factor Xa inhibitor (FXa), is the fourth direct oral anticoagulant (DOAC) approved for clinical use. As the main adverse event is bleeding, it is relevant whether edoxaban has additional effects on platelet function. We aimed to assess in vitro aggregation in patients with atrial fibrillation (AF) receiving edoxaban. We evaluated 20 AF patients treated with edoxaban. We assessed light transmittance platelet aggregation (LTA) with 100 nmol/L γ-thrombin. The LTA was performed at 2 time-points. The thrombin-induced platelet aggregation was significantly lower 2 hours after edoxaban was taken compared to baseline measurement (27.25% ± 30.8% vs. 60.35% ± 33.3%). In addition, we also performed 16 subanalyses in order to identify the differences in the outcome of different comorbidities, age, dosage, liver and kidney function tests, and concomitant treatment. Results of the subgroup analyses were consistent with the findings of the main analysis; there was no apparent heterogeneity across the prespecified subgroups. The thrombin-induced platelet aggregation is reduced in non-valvular AF patients receiving edoxaban.

Thrombin Receptor Agonist Peptide-Induced Platelet Aggregation Is Reduced in Patients Receiving Dabigatran.

The availability of direct oral anticoagulants has caused a paradigm shift in thrombosis management. The direct thrombin inhibitor dabigatran seems to obstruct tenase complex by inhibiting thrombin generated in the initial phase and feed back to the amplification phase of cell-based coagulation reactions. However, it is still not fully understood if and how dabigatran impact platelet function. This observational study aimed to assess in vitro platelet function in patients with atrial fibrillation receiving dabigatran. Platelet aggregability was tested with platelet-rich plasma using platelet aggregometry (PACKS-4 aggregometer). Blood samples were stimulated with thrombin receptor agonist peptide (TRAP; 32 µmol/L). RESULTS: A total of 28 patients with nonvalvular atrial fibrillation were enrolled. The mean age was 71.57 (9.75) years (range: 50-87 years), 16 patients were women, and the mean CHA2DS2VASc score was 3.93 (1.41). All patients began treatment with dabigatran as initial anticoagulant treatment. The minimum term use of dabigatran was 18 days. Dabigatran doses were 110 mg (57.14%) or 150 mg (42.86%) twice daily. The TRAP-induced platelet aggregation was significantly lower 2 hours after taking dabigatran compared to baseline value (79.39 [13.38] vs 90.14 [10.5]). CONCLUSION: The TRAP-induced platelet aggregation was reduced in cardiovascular patients 2 hours after receiving dabigatran. Our findings could have some important clinical implications because platelet aggregation and coagulation cascade are affected at the same time.

Association of Genetic Variability in Selected Genes in Patients With Deep Vein Thrombosis and Platelet Hyperaggregability.

The aim of this study was to evaluate the genetic variability of the selected single nucleotide polymorphisms (SNPs) and examine the association between these SNPs and risk for deep vein thrombosis (DVT) in patients with sticky platelet syndrome (SPS). We examined 84 patients with SPS and history of DVT and 101 healthy individuals. We were interested in 2 SNPs within platelet endothelial aggregation receptor 1 (PEAR1) gene (rs12041331 and rs12566888), 2 SNPs within mkurine retrovirus integration site 1 gene (rs7940646 and rs1874445), 1 SNP within Janus kinase 2 gene (rs2230722), 1 SNP within FCER1G gene (rs3557), 1 SNP within pro-platelet basic protein (rs442155), 4 SNPs within alpha2A adrenergic receptor 2A (ADRA2A; rs1800545, rs4311994, rs11195419, and rs553668), and 1 SNP within sonic hedgehog gene (rs2363910). We identified 2 protective SNPs within PEAR1 gene and 1 risk SNP within ADRA2A gene (PEAR1: rs12041331 and rs12566888; ADRA2A: rs1800545). A haplotype analysis of 4 SNPs within ADRA2A gene identified a risk haplotype aagc ( P = .003). Moreover, we identified 1 protective haplotype within PEAR1 gene (AT, P = .004). Our results support the idea that genetic variability of PEAR1 and ADRA2A genes is associated with platelet hyperaggregability manifested as venous thromboembolism. The study also suggests a possible polygenic type of SPS heredity.

GP6 Haplotype of Missense Variants is Associated with Sticky Platelet Syndrome Manifested by Fetal Loss.

Disequilibrium of hemostasis is central to the pathogenesis of all thromboses, and platelets are essential for primary hemostasis. The platelet membrane glycoprotein receptor is involved in the clot formation in blood; therefore, the changes in related genes could impair platelet aggregation in patients with sticky platelet syndrome (SPS). Patients with SPS who experienced fetal loss were shown to harbor a risk haplotype at GP6 locus. The aim of the study was to examine the genetic linkage of this selected risk haplotype with single nucleotide variations (SNVs) in the coding sequence of the GP6 gene in order to identify possible functional SNVs in association with SPS and fetal loss. A total of 37 patients with SPS manifested fetal loss, and 42 healthy controls were enrolled in the study. The SPS was diagnosed with platelet aggregometry. The SNVs were determined by dideoxy sequencing and high-resolution melting analysis. The missense variations were detected in patients with risk haplotype only. The association analysis showed association of the minor alleles with the SPS manifested by fetal loss as follows-rs1671152 (odds ratio [OR]: 4.667, 95% confidence interval [CI]: 1.462-14.89, P = .006), rs2304167 (OR: 5.085, 95% CI: 1.605-16.10, P = .003), and rs1654416 (OR: 5.085, 95% CI: 1.605-16.10, P = .003). Using the Expectation-Maximization (EM) algorithm, the estimated minor haplotype with predicted protein residue PEAN was significantly associated with the given phenotype (OR: 4.746, 95% CI: 1.486-15.15, P = .005). We have shown that haplotype PEAN associated with SPS and manifested by fetal loss and suggest that the mechanism involved in the action of GPVI has significant effect on GPVI-mediated signal transduction through Syk-phosphorylation.

First Evidence: TRAP-Induced Platelet Aggregation Is Reduced in Patients Receiving Xabans.

The availability of direct oral anticoagulants has caused a paradigm shift in the management of thrombosis. Rivaroxaban and apixaban are 2 direct oral anticoagulants whose target specificity is activated factor X (FXa). However, it is still not fully understood if and how xabans impact platelet function. This observational study aimed to assess the in vitro platelet function in patients with atrial fibrillation receiving xabans. This was a single-center study quantifying platelet aggregation in 41 patients treated with apixaban or rivaroxaban by light transmission aggregometry. The thrombin receptor activating peptide (TRAP)-induced platelet aggregation was significantly lower 2 hours after taking rivaroxaban or apixaban compared to baseline value (56.15% [8.53%] vs 29.51% [12.9%]; P = .000). Moreover, concomitant use of angiotensin-converting enzyme blockers, proton pump inhibitors, and statins reduces the efficiency of xabans. The TRAP-induced platelet aggregation was reduced in patients with cardiovascular disease 2 hours after receiving xabans.

Does type 2 diabetes affect the on-treatment levels of direct oral anticoagulants in patients with atrial fibrillation?

AIMS: Type 2 diabetes (T2D) is connected with several abnormalities in haemostasis; and with higher risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NV-AF). However, it is recently unknown whether T2D affects the activity of direct oral anticoagulants (DOACs). The aim of this study was to determine the impact of T2D on DOACs activity in patients with NV-AF. METHODS: This pilot prospective study enrolled totally 65 patients with NV-AF (20 dabigatran-treated, 110 mg/twice daily; 28 rivaroxaban-treated, 15 mg/daily; 17 apixaban-treated, 5 mg/twice daily). 25 patients had T2D (8 dabigatran-treated, 11 rivaroxaban-treated, and 6 apixaban-treated). DOAC activity was tested with Hemoclot® Thrombin Inhibitor assay in dabigatran-treated patients, and with factor Xa-calibrated anti-Xa chromogenic analysis in rivaroxaban- and apixaban-treated patients prior and two hours after drug administration. RESULTS: There were no significant differences in dabigatran baseline (62.1 ±â€¯8.0 vs. 51.8 ±â€¯38.9 ng/ml, p = .76) and 2-h-post-drug-administration (91.7 ±â€¯57.2 vs. 72.2 ±â€¯33.2 ng/ml, p = .48) activity comparing T2D and non-diabetic patients. Similarly, no significant differences were found in rivaroxaban baseline (35.9 ±â€¯22.5 vs. 55.3 ±â€¯45.1 ng/ml, p = .19) and 2-h-post-drug-administration (145.7 ±â€¯74.1 vs. 202.6 ±â€¯135.0 ng/ml, p = .22) anti-Xa activity. In addition, no significant differences were present in apixaban baseline (96.0 ±â€¯54.5 vs. 63.9 ±â€¯36.8 ng/ml, p = .24) and 2-h-post-drug-administration (151.0 ±â€¯78.3 vs. 151.7 ±â€¯59.1 ng/ml, p = .98) anti-Xa activity between T2D and non-diabetic patients. CONCLUSIONS: This pilot study did not detect differences in DOACs activity according to T2D status in patients with NV-AF.

Soluble P-selectin during a single hemodialysis session in patients with chronic renal failure and erythropoietin treatment.

In several studies, hemodialysis (HD) patients treated with recombinant human erythropoietin (rHuEPO) because of renal anemia showed increased levels of soluble adhesion molecules. The purpose of the study was to investigate the changes of soluble P-selectin (sSELP) and its relationship to platelet activation during a single HD session in patients with long-term rHuEPO treatment. Fifty-two HD patients with chronic renal failure were involved--26 with rHuEPO treatment (EPO group) and 26 without (non-EPO group). Thirty healthy subjects served as the control group. The sSELP, beta-thromboglobulin, and platelet factor 4 plasma levels were measured before and after a single 4-hour HD session on a cuprophane dialyzer. The basal beta-thromboglobulin and platelet factor 4 plasma levels were significantly increased in both HD groups compared with healthy controls but did not change after a single HD session, except for a significant decrease of platelet factor 4 in the non-EPO group. The predialysis sSELP plasma levels did not differ significantly compared with those of the healthy controls, but there was a significant increase of sSELP levels after a single HD session in both groups (EPO, P < .005; non-EPO, P < .05, respectively). These results suppose that the increased sSELP level was released from platelets during the course of a single HD session. The more significant increase of the sSELP plasma levels in EPO group during HD indicates that platelets are more activated in patients with long-term rHuEPO treatment, and this fact could partially explain the suspected tendency for thrombosis in these patients.