Viewpoint: The value of non-criteria antiphospholipid antibodies.

In 2006, at a meeting in Sydney, Australia, consensus was reached by an international group of specialists to establish a number of serological criteria that identify patients with a history of thrombosis or pregnancy complications as having antiphospholipid syndrome (APS). These criteria were originally formulated for research purposes and to compare clinical trials in different centres. However, these same criteria are now generally used and accepted for the diagnosis and treatment of patients. The practice of using these criteria for direct patient care requires that these criteria are based on sound scientific evidence. Indeed, for all the autoantibodies that are officially included in the serological criteria, it has been shown that they induce thrombosis and fetal loss when infused into mice. There are also a number of additional autoantibodies that have been identified in these patients but for these antibodies there was not enough evidence to meet the official APS criteria in 2006. Seventeen years have now passed since the consensus meeting, therefore, this review examines whether additional studies performed with these 'non-criteria' autoantibodies have provided sufficient results to suggest the inclusion of these autoantibodies in the official serological criteria of APS.

A thrombin-driven neural net diagnoses the antiphospholipid syndrome without the need for interruption of anticoagulation.

ABSTRACT: Thrombosis is an important manifestation of the antiphospholipid syndrome (APS). The thrombin generation (TG) test is a global hemostasis assay, and increased TG is associated with thrombosis. APS is currently diagnosed based on clinical and laboratory criteria, the latter defined as anti-cardiolipin, anti-ß2-glycoprotein I antibodies, or lupus anticoagulant (LA). APS testing is often performed after a thrombotic episode and subsequent administration of anticoagulation, which might hamper the interpretation of clotting assays used for LA testing. We set out to develop an artificial neural network (NN) that can diagnose APS in patients who underwent vitamin K antagonist (VKA) treatment, based on TG test results. Five NNs were trained to diagnose APS in 48 VKA-treated patients with APS and 64 VKA-treated controls, using TG and thrombin dynamics parameters as inputs. The 2 best-performing NNs were selected (accuracy, 96%; sensitivity, 96%-98%; and specificity, 95%-97%) and further validated in an independent cohort of VKA-anticoagulated patients with APS (n = 33) and controls (n = 62). Independent clinical validation favored 1 of the 2 selected NNs, with a sensitivity of 88% and a specificity of 94% for the diagnosis of APS. In conclusion, the combined use of TG and NN methodology allowed for us to develop an NN that diagnoses APS with an accuracy of 92% in individuals with VKA anticoagulation (n = 95). After further clinical validation, the NN could serve as a screening and diagnostic tool for patients with thrombosis, especially because there is no need to interrupt anticoagulant therapy.

Crucial roles of red blood cells and platelets in whole blood thrombin generation.

Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C.

BACKGROUND: The acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against ß2-glycoprotein I (ß2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against ß2GPI and prothrombin cause APC resistance is currently unclear. OBJECTIVES: To investigate how anti-ß2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. METHODS: The effects of anti-ß2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. RESULTS: APC resistance was observed in LA-positive patients with anti-ß2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-ß2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-ß2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-ß2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. CONCLUSION: Anti-ß2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

Reversible Platelet Integrin αIIbß3 Activation and Thrombus Instability.

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.

Functional changes in hemostasis during asexual and sexual parasitemia in a controlled human malaria infection.

Decreased platelet count is an early phenomenon in asexual Plasmodium falciparum parasitemia, but its association with acute or long-term functional changes in platelets and coagulation is unknown. Moreover, the impact of gametocytemia on platelets and coagulation remains unclear. We investigated the changes in platelet number and function during early asexual parasitemia, gametocytemia and convalescence in 16 individuals participating in a controlled human malaria infection study, and studied its relationship with changes in total and active von Willebrand factor levels (VWF) and the coagulation system. Platelet activation and reactivity were determined by flow cytometry, and the coagulation system was assessed using different representative assays including antigen assays, activity assays and global functional assays. Platelet count was decreased during asexual blood stage infection but normalized during gametocytemia. Platelet P-selectin expression was slightly increased during asexual parasitemia, gametocytemia and at day 64. In contrast, platelet reactivity to different agonists remained unchanged, except a marked decrease in reactivity to low dose collagen-related peptide-XL. Thrombin generation and antigen assays did not show a clear activation of the coagulation during asexual parasitemia, whereas total and active VWF levels were markedly increased. During gametocytemia and on day 64, the endogenous thrombin potential, thrombin peak and velocity index were increased and prothrombin conversion and plasma prothrombin levels were decreased. We conclude that the decreased platelet count during asexual parasitemia is associated with increased active VWF levels (i.e. endothelial activation), but not platelet hyperreactivity or hypercoagulability, and that the increased platelet clearance in asexual parasitemia could cause spontaneous VWF-platelet complexes formation.

Platelet Activation via Glycoprotein VI Initiates Thrombin Generation: A Potential Role for Platelet-Derived Factor IX?

Collagen triggers coagulation via activation of factor (F) XII. In a platelet-rich environment, collagen can also trigger coagulation independently of FXII. We studied a novel mechanism of coagulation initiation via collagen-dependent platelet activation using thrombin generation (TG) in platelet-rich plasma. Collagen-induced coagulation is minimally affected by active-site inactivated FVIIa, anti-FVII antibodies, or FXIIa inhibition (corn trypsin inhibitor). Activation of platelets via specific glycoprotein (GP) VI agonists initiates TG, FX activation, and fibrin formation. To determine the platelet-derived trigger of coagulation, we systematically reconstituted factor-deficient plasmas with washed platelets. TG triggered by GPVI-activated platelets was significantly affected in FIX- and FVIII-deficient plasma but not in FVII- and FXII-deficient plasma. In a purified system composed of FX and FVIII, we observed that absence of FIX was compensated by GPVI-activated platelets, which could be inhibited by an anti-FIX antibody, suggesting FIXa activity from activated platelets. Furthermore, with the addition of FVIII in FIX-deficient plasma, TG induced by GPVI-activated platelets was restored, and was inhibited by the anti-FIX antibody. In conclusion, GPVI-activated platelets initiate TG, probably via platelet-derived FIXa activity.

Differences in thrombin and plasmin generation potential between East African and Western European adults: The role of genetic and non-genetic factors.

BACKGROUND: Geographic variability in coagulation across populations and their determinants are poorly understood. OBJECTIVE: To compare thrombin (TG) and plasmin (PG) generation parameters between healthy Tanzanian and Dutch individuals, and to study associations with inflammation and different genetic, host and environmental factors. METHODS: TG and PG parameters were measured in 313 Tanzanians of African descent living in Tanzania and 392 Dutch of European descent living in the Netherlands and related to results of a dietary questionnaire, circulating inflammatory markers, genotyping, and plasma metabolomics. RESULTS: Tanzanians exhibited an enhanced TG and PG capacity, compared to Dutch participants. A higher proportion of Tanzanians had a TG value in the upper quartile with a PG value in the lower/middle quartile, suggesting a relative pro-coagulant state. Tanzanians also displayed an increased normalized thrombomodulin sensitivity ratio, suggesting reduced sensitivity to protein C. In Tanzanians, PG parameters (lag time and TTP) were associated with seasonality and food-derived plasma metabolites. The Tanzanians had higher concentrations of pro-inflammatory cytokines, which correlated strongly with TG and PG parameters. There was limited overlap in genetic variation associated with TG and PG parameters between the two cohorts. Pathway analysis of genetic variants in the Tanzanian cohort revealed multiple immune pathways that were enriched with TG and PG traits, confirming the importance of co-regulation between coagulation and inflammation. CONCLUSIONS: Tanzanians have an enhanced TG and PG potential compared to Dutch individuals, which may relate to differences in inflammation, genetics and diet. These observations highlight the importance of better understanding of the geographic variability in coagulation across populations.

Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells.

BACKGROUND: Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. OBJECTIVES: To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. METHODS: The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. RESULTS: Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. CONCLUSIONS: Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

Untargeted Plasma Metabolomics and Gut Microbiome Profiling Provide Novel Insights into the Regulation of Platelet Reactivity in Healthy Individuals.

BACKGROUND: Considerable variation exists in platelet reactivity to stimulation among healthy individuals. Various metabolites and metabolic pathways influence platelet reactivity, but a comprehensive overview of these associations is missing. The gut microbiome has a strong influence on the plasma metabolome. Here, we investigated the association of platelet reactivity with results of untargeted plasma metabolomics and gut microbiome profiling. METHODS: We used data from a cohort of 534 healthy adult Dutch volunteers (the 500 Functional Genomics study). Platelet activation and reactivity were measured by the expression of the alpha-granule protein P-selectin and the binding of fibrinogen to the activated integrin αIIbß3, both in unstimulated blood and after ex vivo stimulation with platelet agonists. Plasma metabolome was measured using an untargeted metabolic profiling approach by quadrupole time-of-flight mass spectrometry. Gut microbiome data were measured by shotgun metagenomic sequencing from stool samples. RESULTS: Untargeted metabolomics yielded 1,979 metabolites, of which 422 were identified to play a role in a human metabolic pathway. Overall, 92/422 (21.8%) metabolites were significantly associated with at least one readout of platelet reactivity. The majority of associations involved lipids, especially members of eicosanoids, including prostaglandins and leukotrienes. Dietary-derived polyphenols were also found to inhibit platelet reactivity. Validation of metabolic pathways with functional microbial profiles revealed two overlapping metabolic pathways ("alanine, aspartate, and glutamate metabolism" and "arginine biosynthesis") that were associated with platelet reactivity. CONCLUSION: This comprehensive overview is an resource for understanding the regulation of platelet reactivity by the plasma metabolome and the possible contribution of the gut microbiota.

Kallikrein augments the anticoagulant function of the protein C system in thrombin generation.

BACKGROUND: Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project. METHODS: Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG. RESULTS: Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 × 10-8 ). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor. CONCLUSIONS: Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis.

Platelet Activation Mechanisms and Consequences of Immune Thrombocytopenia.

Autoimmune disorders are often associated with low platelet count or thrombocytopenia. In immune-induced thrombocytopenia (IIT), a common mechanism is increased platelet activity, which can have an increased risk of thrombosis. In addition, or alternatively, auto-antibodies suppress platelet formation or augment platelet clearance. Effects of the auto-antibodies are linked to the unique structural and functional characteristics of platelets. Conversely, prior platelet activation may contribute to the innate and adaptive immune responses. Extensive interplay between platelets, coagulation and complement activation processes may aggravate the pathology. Here, we present an overview of the reported molecular causes and consequences of IIT in the most common forms of autoimmune disorders. These include idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), drug-induced thrombocytopenia (DITP), heparin-induced thrombocytopenia (HIT), COVID-19 vaccine-induced thrombosis with thrombocytopenia (VITT), thrombotic thrombocytopenia purpura (TTP), and hemolysis, the elevated liver enzymes and low platelet (HELLP) syndrome. We focus on the platelet receptors that bind auto-antibodies, the immune complexes, damage-associated molecular patterns (DAMPs) and complement factors. In addition, we review how circulating platelets serve as a reservoir of immunomodulatory molecules. By this update on the molecular mechanisms and the roles of platelets in the pathogenesis of autoimmune diseases, we highlight platelet-based pathways that can predispose for thrombocytopenia and are linked thrombotic or bleeding events.

Resonance frequency analysis with two different devices after conventional implant placement with ridge preservation: A prospective pilot cohort study.

BACKGROUND: Primary and secondary implant stability is of high importance for survival and success of dental implants in the short and long term. Measurements of implant stability during healing provide the opportunity to monitor the course of the osseointegration process. PURPOSE: To compare implant stability quotient (ISQ) by resonance frequency analysis (RFA), recorded with two different devices after implant placement. MATERIALS AND METHODS: Patients with the need of single tooth extraction in posterior sites of the maxilla and the mandible were treated in a surgical center. All patients received additional augmentation with a bovine bone substitute and platelet-rich fibrin (PRF) after atraumatic tooth extraction. After a healing period of 10 weeks, 28 self-tapping titanium-implants were placed. Implant stability was recorded with two different devices (Osstell and Penguin) at the time of implant insertion (T0), 10 days later (T1), and after 7 (T2), or 17 weeks (T3). RESULTS: No implant was lost, and no postoperative complication occurred during follow-up. Patient cohort comprised 9 female (32.1%) and 19 male patients (67.9%), with a mean age of 52.8 years, 64.3 years, respectively. Mean overall insertion torque was 43.6 Ncm at implant placement with no significant difference between implant location, age, or gender. No patient dropped out. During observation period, a significant increase in mean ISQ was recorded with both devices. Significant positive correlations between insertion torque and ISQ were recorded with both devices at T0, T2, and T3. No significant differences were observed in ISQ-values between both devices, and measuring directions at any point of measurement. CONCLUSIONS: Within the limitations of this cohort study, both devices were suitable for RFA-measurement and revealed comparable results. Due to the cordless design, handling of the Penquin device was more comfortable. Reusability of the Penguin MultiPeg-transducers may offer an additional benefit with regard on ecological aspects.

Deciphered coagulation profile to diagnose the antiphospholipid syndrome using artificial intelligence.

The antiphospholipid syndrome (APS) is diagnosed by the presence of lupus anticoagulant and/or antibodies against cardiolipin or ß2-glycoprotein-1 and the occurrence of thrombosis or pregnancy morbidity. The assessment of overall coagulation is known to differ in APS patients compared to normal subjects. The accelerated production of key factor thrombin causes a prothrombotic state in APS patients, and the reduced efficacy of the activated protein C pathway promotes this effect. Even though significant differences exist in the coagulation profile between normal controls and APS patients, it is not possible to rely on a single test result to diagnose APS. A neural network is a computing system inspired by the human brain that can be trained to distinguish between healthy subjects and patients based on subject specific data. In a first cohort of patients, we developed a neural networking that diagnoses APS. We clinically validated this neural network in a separate cohort consisting of APS patients, normal controls, controls visiting the hospital for other indications and two diseased control groups (thrombosis patients and auto-immune disease patients). The positive predictive value ranged from 62% in the hospital controls to 91% in normal controls and the negative predictive value of the neural network ranged from 86% in the thrombosis control group to 95% in the hospital controls. The sensitivity of the neural network was higher than 90% in all control groups. In conclusion, we developed a neural network that accurately diagnoses APS in the validation cohort. After further clinical validation in newly diagnosed patients, this neural network could possibly be clinically implemented to diagnose APS based on thrombin generation data.

Antiprothrombin antibodies induce platelet activation: A possible explanation for anti-FXa therapy failure in patients with antiphospholipid syndrome?

BACKGROUND: Arterial and venous thrombosis are both common in antiphospholipid syndrome (APS). Recent studies have shown that anti-factor Xa (FXa) therapy in APS patients leads to a greater number of patients with arterial thrombosis than with warfarin. We hypothesize that this may be due to the lowering of prothrombin levels by warfarin. OBJECTIVES: To investigate whether antiprothrombin antibodies induce platelet aggregation and to identify the platelet receptors involved. A second aim was to investigate the effect of reduced prothrombin levels on antiprothrombin antibody-induced platelet aggregation. METHODS: Enzyme-linked immunosorbent assays were performed to measure binding of antiprothrombin antibodies to prothrombin fragment 1+2 and prothrombin. Platelet aggregation assays in washed platelets were performed. FcγRIIA was immunoprecipitated and tyrosine-phosphorylated FcγRIIA was measured by western blot. RESULTS: The antiprothrombin antibodies 28F4 and 3B1 had lupus anticoagulant (LAC) activity and caused platelet aggregation in the presence of Ca2+ and prothrombin. Antiprothrombin antibodies without LAC activity did not activate platelets. Inhibition of Syk and Src kinases and FcγRIIA blocked platelet aggregation. Fab and F(ab')2 fragments of 28F4 were unable to induce platelet aggregation. Immunoprecipitations showed that whole 28F4 immunoglobulin G induced tyrosine phosphorylation of FcγRIIA. Platelet aggregation was significantly reduced when prothrombin levels were reduced from 1 µM to 0.2 µM. CONCLUSIONS: Antiprothrombin antibodies with LAC activity are able to activate platelets via FcγRIIA. Decreased prothrombin levels resulted in less antiprothrombin antibody-mediated platelet aggregation. This may explain the lower incidence of arterial thrombosis in patients treated with warfarin than with anti-FXa therapy.

Long-term treated HIV infection is associated with platelet mitochondrial dysfunction.

HIV infection and antiretroviral therapy have been linked to mitochondrial dysfunction. The role of platelet mitochondrial dysfunction in thrombosis, immunoregulation and age-related diseases is increasingly appreciated. Here, we studied platelet mitochondrial DNA content (mtDNApl) and mitochondrial function in people living with HIV (PLHIV) and related this to platelet function. In a cohort of 208 treated PLHIV and 56 uninfected controls, mtDNApl was quantified, as well as platelet activation, platelet agonist-induced reactivity and inflammation by circulating factors and flow cytometry. In a subgroup of participants, the metabolic activity of platelets was further studied by mitochondrial function tests and the Seahorse Flux Analyzer. PLHIV had significantly lower mtDNApl compared to controls (8.5 copies/platelet (IQR: 7.0-10.7) vs. 12.2 copies/platelet (IQR: 9.5-16.6); p < 0.001), also after correction for age, sex and BMI. Prior zidovudine-use (n = 46) was associated with a trend for lower mtDNApl. PLHIV also had reduced ex vivo platelet reactivity and mean platelet volume compared to controls. MtDNApl correlated positively with both platelet parameters and correlated negatively with inflammatory marker sCD163. Mitochondrial function tests in a subgroup of participants confirmed the presence of platelet mitochondrial respiration defects. Platelet mitochondrial function is disturbed in PLHIV, which may contribute to platelet dysfunction and subsequent complications. Interventions targeting the preservation of normal platelet mitochondrial function may ultimately prove beneficial for PLHIV.

Clinical Relevance of Isolated Lupus Anticoagulant Positivity in Patients with Thrombotic Antiphospholipid Syndrome.

BACKGROUND: Patients positive for all three types of antiphospholipid antibodies (aPLs; triple positivity) have been identified for having a high risk for thrombotic events. However, the clinical significance of isolated lupus anticoagulant (LAC) positivity is debated. OBJECTIVES: To investigate the clinical relevance of isolated LAC. METHODS: A total of 456 patients were enrolled in this study; 66 antiphospholipid syndrome patients and 390 control patients. The control group consisted of autoimmune patients (n = 91), patients with thrombosis but without aPLs (n = 127), and normal controls (n = 172). LAC, anticardiolipin (anti-CL), and anti-ß2 glycoprotein I (anti-ß2GPI) immunoglobulin G (IgG) and immunoglobulin M (IgM) were determined according to the International Society on Thrombosis and Haemostasis (ISTH) guidelines. Anti-CL and anti-ß2GPI were measured by four different solid-phase platforms to overcome variability between test systems. The noncriteria IgA anti-CL and anti-ß2GPI, antidomain I of ß2GPI IgG, and antiphosphatidylserine/prothrombin antibodies (anti-PS/PT) IgG and IgM were detected according to the ISTH guidelines for solid-phase assays. RESULTS: In total, 70 patients were positive for LAC, of which 44 were negative for both anti-ß2GPI and anti-CL antibodies. We found that isolated LAC proved to be strongly associated with vascular thrombosis (odds ratio [OR]: 7.3; 95% confidence interval [CI]: 3.3-16.1), even better than triple-positive samples (OR: 4.3; 95% CI: 1.6-12.2). The titers of the anti-PS/PT IgG and IgM were significantly higher in triple-positivity samples compared with samples with isolated LAC positivity. The majority of single LAC positives were anti-PS/PT-negative. We observed that LAC positivity was weaker in isolated LAC-positive patients compared with LAC activity in triple-positive patients. CONCLUSION: Isolated LAC was highly associated with thrombosis. The presence of anti-PS/PT antibodies could not explain LAC positivity in isolated LAC. Isolated LAC showed a weaker LAC activity compared with triple-positive patients.

Anti-ß2-glycoprotein I and anti-prothrombin antibodies cause lupus anticoagulant through different mechanisms of action.

BACKGROUND: The presence of lupus anticoagulant (LA) is an independent risk factor for thrombosis. This laboratory phenomenon is detected as a phospholipid-dependent prolongation of the clotting time and is caused by autoantibodies against beta2-glycoprotein I (ß2GPI) or prothrombin. How these autoantibodies cause LA is unclear. OBJECTIVE: To elucidate how anti-ß2GPI and anti-prothrombin antibodies cause the LA phenomenon. METHODS: The effects of monoclonal anti-ß2GPI and anti-prothrombin antibodies on coagulation were analyzed in plasma and with purified coagulation factors. RESULTS: Detection of LA caused by anti-ß2GPI or anti-prothrombin antibodies required the presence of the procofactor factor V (FV) in plasma. LA effect disappeared when FV was replaced by activated FV (FVa), both in a model system and in patient plasma, although differences between anti-ß2GPI and anti-prothrombin antibodies were observed. Further exploration of the effects of the antibodies on coagulation showed that the anti-ß2GPI antibody attenuated FV activation by activated faxtor X (FXa), whereas the anti-prothrombin antibody did not. Binding studies showed that ß2GPI--antibody complexes directly interacted with FV with high affinity. Anti-prothrombin complexes caused the LA phenomenon through competition for phospholipid binding sites with coagulation factors as reduced FXa binding to lipospheres was observed with flow cytometry in the presence of these antibodies. CONCLUSION: Anti-ß2GPI and anti-prothrombin antibodies cause LA through different mechanisms of action: While anti-ß2GPI antibodies interfere with FV activation by FXa through a direct interaction with FV, anti-prothrombin antibodies compete with FXa for phospholipid binding sites. These data provide leads for understanding the paradoxical association between thrombosis and a prolonged clotting time in the antiphospholipid syndrome.