Surgery in rare bleeding disorders: the prospective MARACHI study.

Background: Despite the wide use of bleeding scores and the reliability of clotting factor level measurement, bleeding risk stratification before surgery remains challenging in patients with rare inherited bleeding disorders. Objectives: This multicenter observational prospective study assessed in patients with rare coagulation factor deficiency, the perioperative hemostatic management choices by hemostasis experts and the bleeding outcomes after surgery. Methods: One hundred seventy-eight patients with low coagulation activity level (factor [F] II, FV, combined FV-FVIII, FVII, FX, or FXI <50%) underwent 207 surgical procedures. The bleeding outcome, Tosetto's bleeding score, and perioperative hemostatic protocols were collected. Results: Among the 81 procedures performed in patients with severe factor deficiency (level ≤10%), 27 were done without factor replacement (including 6 in patients at high bleeding risk), without any bleeding event. Factor replacement therapy was used mainly for orthopedic procedures. In patients with mild deficiency, 100/126 surgical procedures were carried out without perioperative hemostatic treatment. In patients with FVII or FXI deficiency, factor replacement therapy was in function of the procedure, bleeding risk, and to a lesser extent previous bleeding history. Tranexamic acid was used in almost half of the procedures, particularly in case of surgery in tissues with high fibrinolytic activity (76.8%). Conclusions: The current perioperative hemostatic management of patients with rare bleeding disorders appears to be adapted. Among the 207 procedures, only 6 were associated with excessive bleeding. Our findings suggest that rather than the bleeding score, factor level and surgery type are the most relevant criteria for perioperative factor replacement therapy.

Severe thrombophilia in a factor V-deficient patient homozygous for the Ala2086Asp mutation (FV Besançon).

BACKGROUND: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions. OBJECTIVE: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. METHODS/RESULTS: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5-fold) unfavorable kinetic parameters (Km , Vmax ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S. CONCLUSIONS: FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.

Laboratory Techniques Used to Diagnose Constitutional Platelet Dysfunction.

Platelets play a major role in primary hemostasis, where activated platelets form plugs to stop hemorrhaging in response to vessel injuries. Defects in any step of the platelet activation process can cause a variety of platelet dysfunction conditions associated with bleeding. To make an accurate diagnosis, constitutional platelet dysfunction (CPDF) should be considered once von Willebrand disease and drug intake are ruled out. CPDF may be associated with thrombocytopenia or a genetic syndrome. CPDF diagnosis is complex, as no single test enables the analysis of all aspects of platelet function. Furthermore, the available tests lack standardization, and repeat tests must be performed in specialized laboratories especially for mild and moderate forms of the disease. In this review, we provide an overview of the laboratory tests used to diagnose CPDF, with a focus on light transmission platelet aggregation (LTA), flow cytometry (FC), and granules assessment. Global tests, mainly represented by LTA, are often initially performed to investigate the consequences of platelet activation on platelet aggregation in a single step. Global test results should be confirmed by additional analytical tests. FC represents an accurate, simple, and reliable test to analyze abnormalities in platelet receptors, and granule content and release. This technique may also be used to investigate platelet function by comparing resting- and activated-state platelet populations. Assessment of granule content and release also requires additional specialized analytical tests. High-throughput sequencing has become increasingly useful to diagnose CPDF. Advanced tests or external research laboratory techniques may also be beneficial in some cases.

Receptor for Advanced Glycation End Products is Involved in Platelet Hyperactivation and Arterial Thrombosis during Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is associated with a high cardiovascular mortality due to increased rates of vascular lesions and thrombotic events, as well as serum accumulation of uremic toxins. A subgroup of these toxins (advanced glycation end products [AGEs] and S100 proteins) can interact with the receptor for AGEs (RAGE). In this study, we analyzed the impact of CKD on platelet function and arterial thrombosis, and the potential role of RAGE in this process. METHODS: Twelve weeks after induction of CKD in mice, platelet function and time to complete carotid artery occlusion were analyzed in four groups of animals (sham-operated, CKD, apolipoprotein E [Apoe]-/-, and Apoe-/-/Ager-/- mice). RESULTS: Analysis of platelet function from whole blood and platelet-rich plasma showed hyperactivation of platelets only in CKD Apoe-/- mice. There was no difference when experiments were done on washed platelets. However, preincubation of such platelets with AGEs or S100 proteins induced RAGE-mediated platelet hyperactivation. In vivo, CKD significantly reduced carotid occlusion times of Apoe-/- mice (9.2 ± 1.1 vs. 11.1 ± 0.6 minutes for sham, p < 0.01). In contrast, CKD had no effect on occlusion times in Apoe-/-/Ager-/- mice. Moreover, carotid occlusion in Apoe-/- CKD mice occurred significantly faster than in Apoe-/-/Ager-/- CKD mice (p < 0.0001). CONCLUSION: Our results show that CKD induces platelet hyperactivation, accelerates thrombus formation in a murine model of arterial thrombosis, and that RAGE deletion has a protective role. We propose that RAGE ligands binding to RAGE is involved in CKD-induced arterial thrombosis.

Emicizumab treatment: Impact on coagulation tests and biological monitoring of haemostasis according to clinical situations (BIMHO group proposals).

Emicizumab, a bispecific humanised monoclonal antibody restoring to some extent the function of activated FVIII deficient in haemophilia A, represents a major therapeutic advance in the management of haemophilia A patients. No dosage adjustment is required, which leads to a major change for patients used to regular biological monitoring which is particularly burdensome in the case of substitution therapy. In some circumstances, such as before an invasive procedure or in case of bleeding, biological monitoring will be necessary and emicizumab's interference with haemostasis tests, particularly those based on an activated partial thromboplastin times (aPTT), must be known to best interpret the tests and to select the most appropriate methods to guide therapy. The normalisation of aPTT in patients treated with emicizumab is not sufficient to consider haemostasis as normalised. In the event of administration of FVIII to a patient receiving emicizumab, the determination of FVIII should use a chromogenic method using non-human reagents. Coagulation global tests have been proposed to evaluate the biological response when using bypassing agents in patients treated with emicizumab, but the usefulness must be confirmed. The French group BIMHO presents proposals for biological monitoring of a patient treated with emicizumab according to clinical situations.

Elastin-derived peptides are new regulators of thrombosis.

OBJECTIVE: Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis. APPROACH AND RESULTS: We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen. CONCLUSIONS: These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.

Performance of two new automated assays for measuring von Willebrand activity: HemosIL AcuStar and Innovance.

The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20-30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the "gold standard" VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.

Spectrum of the mutations in Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb-IX-V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbß), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia.

A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.

Anticoagulant properties of the anti-inflammatory cytokine IL-10 in a factor Xa-activated human monocyte model.

BACKGROUND: Monocytes and factor Xa (FXa) are procoagulant agents implicated in the physiopathological processes of atherosclerosis and thrombosis. OBJECTIVE: we evaluated the anticoagulant effect of the anti-inflammatory cytokine IL-10 on an FXa-activated human monocyte (Hu-monocyte) procoagulant phenotype. METHODS: Hu-monocytes were purified by elutriation and activated by FXa. The effect of IL-10 was assessed by means of a 2 h pre-incubation step with recombined human IL-10 (0.5 and 1 ng/mL). Real-time RT-PCR and Western blotting were used to evaluate the effect of IL-10 on tissue factor (TF) mRNA and protein synthesis. A thrombin generation (TG) assay was used as a functional test to assess the effect of IL-10 on TF-dependent TG. RESULTS: we showed that IL-10 inhibited both TFmRNA and TF protein expression in a dose-dependant manner.We showed, as a functional consequence, that IL-10 inhibited Hu-monocyte-triggered TG and that this inhibition was concentration-dependant, and significant for all TG phases. The rate index of the propagation phase (rate index) was the most sensitive parameter while the endpoint of TG decay (S-tail) and the endogenous thrombin potential (ETP) were the least sensitive (inhibition of 80, 40 and 30% respectively). The IL-10 pattern of TG inhibition was similar to TF-Ab-induced inhibition: IC(50) were not reached by ETP and S-tail, and the lowest IC(50) values were reached by the rate index (0.61 ± 0.12 ng/mL and 1.87 ± 0.35 µg/mL respectively). CONCLUSION: the anticoagulant effect of the anti-inflammatory cytokine IL-10 in an FXa-activated Hu-monocyte model is an additional illustration of the cross-talk between inflammation and coagulation, opening new approaches in the field of arteriosclerosis and thrombosis.

IL-10 modulates fondaparinux inhibition of monocyte-induced thrombin generation.

In addition to its established immuno-regulating capacity, the anti-inflammatory cytokine interleukin (IL)-10 exerts direct effects on coagulation. IL-10 down regulates the expression of tissue factor (TF) and thrombin generation (TG). Thus, we hypothesised that IL-10 could enhance the effect of anticoagulants. To evaluate in vitro the potential additive effect of IL-10 on fondaparinux-induced anticoagulation. Human monocytes were purified by elutriation, and were activated by factor Xa (FXa). Real-time RT-PCR and Western blotting were used to evaluate FXa-induced TF synthesis. TG test was used as a functional test to assess TF-dependent monocyte procoagulation, and to evaluate the effects of IL-10 (200 and 500 pg/ml) and fondaparinux (0.0, 0.1, 0.4, 0.7 and 1.2 µg/ml), separately and in combination. We confirmed that FXa induced TF mRNA and protein synthesis by monocyte in a concentration dependent manner. We showed that FXa-activated monocytes triggered TG via TF expression. We reported that IL-10 inhibited TG with a marginal effect seen at 200 pg/ml. Results with fondaparinux showed a concentration-dependent TG inhibition. The combination of IL-10 and fondaparinux effects demonstrated that IL-10: (i) potentiates the inhibitory effect of fondaparinux on TG by 10-30%, and (ii) dramatically modifies fondaparinux IC50 for each TG parameter. IL-10 enhances in vitro the extent of anticoagulation induced by fondaparinux.

Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation.

Anticoagulants, including unfractionated heparin (UFH), enoxaparin and fondaparinux, are approved drugs in acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high tissue factor (TF) levels, which in turn triggers thrombin generation. The objective of our study is to compare the in-vitro inhibitory effect of UFH, enoxaparin and fondaparinux in monocytes and MMP models. Human-elutriated monocytes were activated for 5 and 18 h by lipopolysaccharide to obtain activated monocytes (ac-M) or MMPs, respectively. Thrombin generation inhibition was assessed using ac-M or MMPs mixed with platelet-poor plasma containing increased concentrations of anticoagulants. Thrombin generation inhibition was dose-dependent with a differential effect according to the drug: the highest for UFH, the lowest for fondaparinux. Rate index was the most sensitive parameter. For fondaparinux, its IC50 values (anti-Xa IU/ml) were 0.59±0.05 for ac-M and 0.17±0.03 for MMPs. For enoxaparin, rate index IC50 values were 0.27±0.03 for ac-M and 0.19±0.02 for MMPs. Our data support the notion that cell-induced thrombin generation assay may be a reliable alternative to anti-Xa assessment in determining patient anticoagulation level.

Differential inhibitory effect of fondaparinux on the procoagulant potential of intact monocytes and monocyte-derived microparticles.

Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 106 and 1 × 106/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 µg/ml). Without fondaparinux, 0.2 × 106 ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 µg/ml for ac-M, 0.20 ± 0.03 µg/ml for MMPs, and 0.22 ± 0.02 µg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 µg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.

AlphaIIbbeta3 integrin: new allelic variants in Glanzmann thrombasthenia, effects on ITGA2B and ITGB3 mRNA splicing, expression, and structure-function.

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin alphaIIbbeta3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North-African and Asian were characterized. Promoter and exon sequences of alphaIIb and beta3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The alphaIIb p.S926L, p.V903F, and beta3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS-7 cells by altering the alphaIIb or the beta3 subunit structure. As shown by free energy analyses applied on the resolved structure of alphaIIbbeta3 and structural modeling of the mutant, the p.K253M substitution of beta3 helped to define a key role of the K253 in the interaction of the alphaIIb beta-propeller and the beta3 beta-I domains. finally, the alphaIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of alphaIIb for the alphaIIbbeta3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect.

Is flow cytometry accurate enough to screen platelet autoantibodies?

BACKGROUND: The diagnosis of immune thrombocytopenic purpura (ITP) is a diagnosis of exclusion, as stated by international guidelines. Nevertheless, the assessment of platelet (PLT) antibodies has been reported as helpful for the diagnosis and the follow-up of ITP patients. PLT antibodies are detected by highly specialized assays, such as monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test. Flow cytometry for PLT-associated immunoglobulin G (PAIgG) detection has been described more recently. This study was meant to evaluate the utility of flow cytometry to screen accurately patients needing further MAIPA testing. STUDY DESIGN AND METHODS: PAIgG, PAIgM, and PAIgA were determined in 107 consecutive patients and in 147 healthy controls in parallel. MAIPA testing was performed in all patients. The accuracy of flow cytometry was assessed with a receiver operating characteristics (ROC) curve analysis versus MAIPA. RESULTS: MAIPA assay found PLT-specific IgG in 27 patients (25%). The ROC curve analysis showed that no false-negative result in flow cytometry was obtained for a mean fluorescence intensity (MFI) cutoff of 0.2. With this cutoff, PAIgG were positive in 61 patients (57%). In this series, MAIPA was unnecessary in 42 percent of patients (corresponding to true-negative results). When MAIPA was positive, PAIgM values ranged from 0.1 to 1.0, and PAIgA from 0.1 to 2. CONCLUSION: Flow cytometry for PAIgG assessment may be used to accurately decide whether or not MAIPA must be subsequently performed. In this series, MAIPA was unnecessary in 42 percent of patients. Moreover, PAIgM results suggested that its determination combined with PAIgG may be of interest in ITP investigation.

A case control study of deep venous thrombosis in relation to factor V G1691A (Leiden) and A4070G (HR2 Haplotype) polymorphisms.

Activated protein C resistance (APCR) is a significant risk factor for venous thromboembolism (VTE), with the factor V (FV) G1691A (Leiden) mutation accounting for the majority of inherited APCR cases. An additional FV polymorphism, A4074G (FV-HR2), reportedly increased VTE risk by some, but not all groups. We determined the prevalence of FV-Leiden and FV-HR2 SNPs in 126 patients with deep venous thrombosis (DVT), and 197 control subjects. Frequencies of FV-Leiden A and HR2 G alleles, together with FV-Leiden G/A and A/A (but not HR2 A/G) genotypes were significantly higher among patients. While no significant linkage disequilibrium was noted between FV 1691A and 4070G or A alleles, significantly higher prevalence of single-mutant 1691G/4070G and 1691A/4070A haplotypes were seen in patients. FV Leiden and FV HR2 haplotype are independent risk factors for DVT, and their coinheritance does not seem to increase significantly DVT risk imparted by either.