Elevated factor XIa as a modulator of plasma fibrin clot properties in coronary artery disease.

INTRODUCTION: Patients with coronary artery disease (CAD) display a prothrombotic fibrin clot phenotype, involving low permeability and resistance to lysis. The determinants of this phenotype remain elusive. Circulating tissue factor (TF) and activated factor XI (FXIa) are linked to arterial thromboembolism. We investigated whether detectable active TF and FXIa influence fibrin clot properties in CAD. METHODS: In 118 CAD patients (median age 65 years, 78% men), we assessed Ks, an indicator of clot permeability, and clot lysis time (CLT) in plasma-based assays, along with the presence of active TF and FXIa. We also analysed proteins involved in clotting and thrombolysis, including fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable thrombolysis inhibitor (TAFI). During a median 106 month (interquartile range 95-119) follow-up, myocardial infarction (MI), stroke, systemic thromboembolism (SE) and cardiovascular (CV) death were recorded. RESULTS: Circulating TF and FXIa, detected in 20.3% and 39.8% of patients, respectively, were associated with low Ks and prolonged CLT. Solely FXIa remained an independent predictor of low Ks and high CLT on multivariable analysis. Additionally, fibrinogen and PAI-1 were associated with low Ks, while PAI-1 and TAFI-with prolonged CLT. During follow-up low Ks and prolonged CLT increased the risk of MI and the latter also a composite endpoint of MI, stroke/SE or CV death. CONCLUSIONS: To our knowledge, this study is the first to show that circulating FXIa is associated with prothrombotic fibrin clot properties in CAD, suggesting additional mechanisms through which FXIa inhibitors could act as novel antithrombotic agents in CAD.

Activated factor XI is associated with increased factor VIIa - Antithrombin complexes in stable coronary artery disease: Impact on cardiovascular outcomes.

BACKGROUND: Coronary artery disease (CAD) is associated with a prothrombotic tendency including increased factor (F) VIIa-antithrombin (FVIIa-AT) complexes, a measure of tissue factor (TF) exposure, and activated FXI (FXIa). We investigated whether increased FVIIa-AT complexes are associated with FXIa and active TF and if major adverse clinical outcomes are predicted by the complexes in CAD. METHODS: In 120 CAD patients, we assessed FVIIa-AT complex concentrations and the presence of circulating FXIa and active TF. Levels of 8-iso-prostaglandin F2α (8-iso-PGF2α), interleukin-6, high-sensitivity C reactive protein, prothrombin fragment 1 + 2, and free Tissue Factor Pathway Inhibitor were determined. Myocardial infarction (MI), ischemic stroke, systemic thromboembolism (SE), and cardiovascular (CV) death were recorded separately and as a composite endpoint, during follow-up. RESULTS: FVIIa-AT complexes were positively associated with current smoking and multivessel CAD. Elevated FVIIa-AT complexes characterized patients with circulating FXIa and/or active TF in association with increased plasma isoprostanes but not with thrombin generation or inflammatory markers. During a median follow-up of 106 months (interquartile range 95-119), high baseline levels of FVIIa-AT complexes predicted ischemic stroke/SE (HR 4.61 [95% CI 1.48-18.42]) and a composite endpoint of MI, stroke/SE, and CV death (HR 7.47 [95% CI 2.81-19.87]). CONCLUSIONS: This study is the first to show that high FVIIa-AT complexes characterize advanced CAD patients with detectable FXIa and active TF, which is, in part, driven by oxidative stress. High FVIIa-AT complexes were associated with the risk of ischemic stroke/SE during long-term follow-up, highlighting the need for effective antithrombotic agents in CAD.

Predictors of neutrophil extracellular traps markers in type 2 diabetes mellitus: associations with a prothrombotic state and hypofibrinolysis.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is associated with a hypercoagulable state and increased neutrophil extracellular traps formation (NETosis). We investigated predictors of NETosis and cell death markers in circulating blood and their association with a prothrombotic state in T2DM. METHODS: In a cross-sectional study involving 113 T2DM patients aged 63.7 ± 8.2 years, we investigated citrullinated histone H3 (H3Cit), cell-free deoxyribonucleic acid (cfDNA), myeloperoxidase, neutrophil elastase, and inflammation markers, along with thrombin generation (TG), plasma clot lysis time (CLT), clot permeability (Ks) and fibrinolysis inhibitors. RESULTS: On multivariate logistic regression analysis adjusted for age and gender, predictors of high H3Cit (≥ 7.36 ng/mL, upper quartile) were: glycated hemoglobin (HbA1c) ≥ 7.0% and interleukin-6. Interleukin-6 was also found to be a predictor of high cfDNA (≥ 2.84 µg/mL, upper quartile) along with glucose. Citrullinated histone H3 and cfDNA correlated positively with CLT and inversely with Ks, while TG associated solely with cfDNA. These associations were not seen with myeloperoxidase and neutrophil elastase. Patients with previous myocardial infarction (n = 21, 18.6%) had higher H3Cit (+108%, p < 0.001) and cfDNA (+45%, p = 0.022). On multivariable analysis adjusted for potential confounders, H3Cit and cfDNA, along with plasminogen activator inhibitor-1 and concomitant cardiovascular disease, were predictors of CLT. Citrullinated histone H3 alone was a predictor of Ks and only cfDNA was a predictor of peak thrombin generated. CONCLUSIONS: In T2DM, NETosis detectable in circulating blood is associated with inflammatory state and a prothrombotic state, especially hypofibrinolysis.

Continuous thrombin generation in whole blood: New applications for assessing activators and inhibitors of coagulation.

Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo. We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ±â€¯5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders.

Issues complicating precision dosing for factor VIII prophylaxis.

We previously showed that personalizing prophylaxis on the basis of an individual's pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis. A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains. Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing. Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis.

Activation of blood coagulation and thrombin generation in acute ischemic stroke treated with rtPA.

The impact of thrombolysis with recombinant tissue plasminogen activator (rtPA) on blood coagulation in acute ischemic stroke (AIS) patients is not completely understood. We studied the effect of thrombolysis on the thrombin generation (TG) profile as well as coagulant activity of activated factors IX (FIXa), XI (FXIa) and tissue factor (TF) in AIS patients. In a case-control study, TG parameters as well as FIXa, FXIa and TF levels were assessed in 95 AIS patients, including individuals receiving rtPA treatment within 4.5 h since AIS onset (n = 71, 74.7%) and those ineligible for thrombolysis (n = 24, 25.3%). Blood samples were collected at baseline and after 24 h since admission. The two groups were similar with regard to demographics and clinical factors. In thrombolysed patients, all TG parameters measured after 24 h were markedly decreased, with strongest impact on lag time (LT), when compared with the baseline values (81.3% longer LT, p < 0.0001), as well as when compared to the non-thrombolysed group (86% longer LT, p = 0.002). In non-thrombolysed AIS patients the TG remained unaltered. Logistic regression adjusted for potential confounders showed that high baseline ETP value (the top quartile) was solely predicted by the presence of circulating FIXa, whereas after 24 h FXIa predicted high ETP in the subgroup of thrombolysed and in all AIS patients. Thrombolysis in AIS patients markedly attenuates the TG. Elevated FXIa contributes to thrombin formation capacity after 24 h, highlighting a role of this factor in the regulation of blood coagulation in AIS.

Procoagulant activity in stored units of red blood cells.

The procoagulant activity (PA) of stored units of red blood cells (RBC) increases over time, which is related to the expression/exposure of tissue factor (TF). However, there is a discrepancy between the TF measured and changes in PA observed, suggesting that other blood components contribute to this activity. Our goal was to evaluate changes in PA of stored RBCs and to determine possible contributors to it. RBC units from 4 healthy donors were prepared and stored at 4 °C. On selected days, RBC aliquots were reconstituted with autologous plasma and tested in the thromboelastography assay. Corresponding supernatants were tested in a clotting assay. For all donors, the clotting time (CT) of reconstituted RBC units decreased from ∼3000-4000s on day 1 to ∼1000-1600s on day 30, with the most dramatic changes occurring between days 1 and 5. Anti-TF antibody slightly prolonged the CT. The concentration of TF did not change significantly over time and was within the range of 0.3-2.3 pM. Bovine lactadherin (LTD) prolonged the CT of the RBC (by 2.4-3.4-fold in days 3-5 and by 1.3-1.8-fold at day 30). Anti-TF antibody together with LTD had a cumulative effect on the CT prolongation. CT of supernatants responded to both anti-TF and anti-FXIa antibodies. Three contributors to the PA of stored RBC were identified, i.e. FXIa in solution and phosphatidylserine and TF exposed on blood cells and microparticles. Failure of LTD and antibodies to completely eliminate PA suggests that other components of blood could contribute to it.

Platelets do not express the oxidized or reduced forms of tissue factor.

BACKGROUND: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms. METHODS: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results. RESULTS: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography. CONCLUSION AND SIGNIFICANCE: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.

Disulfide reduction abolishes tissue factor cofactor function.

BACKGROUND: Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186-Cys209, has been hypothesized to be essential for an allosteric "decryption" phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties. METHODS: The status of disulfides in recombinant TF1-263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function. RESULTS: In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function. CONCLUSION: The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis. GENERAL SIGNIFICANCE: Results of this study advance our knowledge on TF structure/function relationships.

Comparison of natural and recombinant tissue factor proteins: new insights.

Tissue factor (TF), an initiator of blood coagulation in vivo, is expressed in a variety of cells. Sufficient natural TF has been isolated to clone and express recombinant proteins ranging from full-length TF to its extracellular domain. Because of the limited availability of natural TF, recombinant proteins have been used as surrogates. Despite the differences in their post-translational modifications, it has been accepted that membrane-anchored recombinant TFs are quite similar to the natural TF. Recent studies, however, have shown that post-translational modifications play an important role in TF-triggered thrombin generation.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, and peptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

Differences in the fractional abundances of carbohydrates of natural and recombinant human tissue factor.

BACKGROUND: Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371-3382). METHODS: In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. RESULTS AND CONCLUSIONS: Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. CONCLUSION: These observations may attribute to the difference in the ability of TF-FVIIa complex to activate factor X (FX). GENERAL SIGNIFICANCE: Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF.

Cellular regulation of blood coagulation: a model for venous stasis.

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R(506) and R(306). Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R(306) was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

Active tissue factor and activated factor XI in patients with acute ischemic cerebrovascular events.

BACKGROUND: Elevated factor (F)XI and tissue factor (TF) have been reported to occur in patients with acute ischemic stroke (AIS). We sought to investigate whether circulating activated FXI (FXIa) and TF on admission can predict clinical outcomes in patients with acute cerebrovascular events. MATERIALS AND METHODS: In the observational study, we evaluated 205 consecutive patients aged 70 years or less within the first 72 h of acute event, including 140 with AIS and 65 with transient ischemic attack (TIA). Plasma TF and FXIa activity were determined on admission in clotting assays by measuring the response to inhibitory monoclonal antibodies. RESULTS: Active TF and FXIa activity were detected in 58 (28·9%) and 132 (64·4%) patients on admission, respectively. Active TF was detected in 45 of the 136 AIS patients with available TF levels (33·1%) and 13 of the 65 patients with acute TIA (20%; 0·05). Corresponding values for FXIa were 99 of the 140 (70·7%) and 33 of the 65 (50·8%; P= 0·006), respectively. Patients with detectable TF were more frequently women and hypertensive, while subjects with detectable FXIa had more often diabetes and higher levels of fibrinogen, C-reactive protein and interleukin-6 (all P < 0·05). Patients with detectable FXIa but not TF had higher National Institutes of Health Stroke Scale score, higher modified Rankin scale score and lower Barthel Index at discharge (all P < 0·05). CONCLUSIONS: Circulating active TF and FXIa occur frequently in acute cerebrovascular ischemic events. Active FXIa in plasma might be useful as a novel risk marker of worse functional outcomes in patients with acute cerebrovascular events.

Active factor XI is associated with the risk of cardiovascular events in stable coronary artery disease patients.

BACKGROUND AND AIMS: Tissue factor (TF) and activated factor XI (FXIa) have been associated with acute coronary syndrome, ischemic stroke and venous thromboembolism. Their predictive value in stable coronary artery disease (CAD) is unclear. We investigated whether active TF and FXIa were associated with clinical outcomes in CAD patients in long-term observation. METHODS: In 124 stable patients with multivessel CAD, we assessed the presence of circulating, active TF and FXIa by measuring a response of thrombin generation to respective inhibitory antibodies. We recorded the composite endpoint of myocardial infarction (MI), stroke, systemic thromboembolism and cardiovascular death during follow-up (median 106 months, interquartile range 95-119). RESULTS: Circulating FXIa and active TF were detected in 40% and 20.8% of the 120 patients (aged 65.0 [57.0-70.3] years, men, 78.3%), who completed follow-up. The composite endpoint occurred more frequently in patients with detectable active TF and FXIa present at baseline (hazard ratio [HR] 4.02, 95% confidence interval [CI] 2.26-7.17, p < 0.001 and HR 6.21, 95% CI 3.40-11.40, p < 0.001, respectively). On multivariate analysis FXIa, but not active TF, was an independent predictor of the composite endpoint, as well as MI, stroke/systemic thromboembolism, and cardiovascular death, when analyzed separately. CONCLUSIONS: To our knowledge, this study is the first to show that circulating FXIa predicts arterial thromboembolic events in advanced CAD, supporting a growing interest in FXIa inhibitors as novel antithrombotic agents.

Active FXI Can Independently Predict Ischemic Stroke in Anticoagulated Atrial Fibrillation Patients: A Cohort Study.

BACKGROUND: Atrial fibrillation (AF) is associated with a prothrombotic state. Presence of active tissue factor (TF), activated factor IX (FIXa) and FXIa in circulating blood contributes to thrombosis. We investigated a prognostic value of these factors in AF patients. METHODS: In this cohort study, 284 AF patients (aged 63.3 ± 8.8 years) treated with oral anticoagulants were enrolled. Plasma levels of active coagulation factors were evaluated using thrombin generation assay. Concentrations of fibrinogen, D-dimer, interleukin-6 (IL-6), and endothelial damage markers, including von Willebrand factor (VWF) and soluble (s)E-selectin, were also measured. Ischemic stroke and cardiovascular death, analyzed separately or as a composite endpoint, were recorded during a mean follow-up of 47 months. RESULTS: Cerebrovascular events were observed in 20 patients (1.8%/year) who had at baseline higher fibrinogen, D-dimer, and VWF levels. Active TF and FXIa at enrollment were detectable in 12 (60%) and 15 (75%) patients who experienced ischemic stroke during follow-up. The composite endpoint observed in 23 patients (2.1%/year) was associated with increased concentrations of the above laboratory variables, along with 26% higher IL-6 levels. sE-selectin did not differ between the studied groups. On multivariable regression analysis, advanced age, anticoagulation discontinuation, and detectable FXIa, but not active TF, independently predicted the composite endpoint. No associations of FIXa with the study endpoints were observed. CONCLUSION: FXIa present in circulating blood is associated with increased risk of ischemic stroke and cardiovascular death in anticoagulated AF patients during long-term follow-up. FXIa inhibition could be useful in cardiovascular prevention in AF beyond the current oral anticoagulation.

Carbohydrates and activity of natural and recombinant tissue factor.

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF.factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (V(max)) of the complex increased in the order rTF(1-243) (Escherichia coli) < rTF(1-263) (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K(m) of FX for rTF(1-263)-FVIIa remained unchanged after deglycosylation, whereas the k(cat) decreased slightly. A pronounced decrease, 4-fold, in k(cat) was observed for pTF.FVIIa upon deglycosylation, whereas the K(m) was minimally altered. The parameters of FX activation by both rTF(1-263D)-FVIIa and pTF(D)-FVIIa were identical and similar to those for rTF(1-243)-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF(1-263) and pTF. The carbohydrates of rTF(1-263) contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.

Quantitation of anti-factor VIII antibodies in human plasma.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (+/-1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)- coupled microspheres and probed with mouse anti-human Ig-R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 +/- 0.8 nM), 11 BU/mL (214.3 +/- 7.1 nM), 106 BU/mL (2209.4 +/- 84.9 nM), 140 BU/mL (2417.7 +/- 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti-human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

Platelet-white blood cell (WBC) interaction, WBC apoptosis, and procoagulant activity in stored red blood cells.

BACKGROUND: Nonleukoreduced units of red blood cells (RBCs) contain activated platelets (PLTs) that interact with white blood cells (WBCs) and may promote inflammation and thrombosis in the recipient. The aim of this study was to characterize PLT-WBC interactions (PLT-WBC aggregates [PLAs]), WBC apoptosis, WBC death, and the development of procoagulant activity in RBCs during storage. STUDY DESIGN AND METHODS: RBCs were prepared from volunteer donor blood and stored. Samples were analyzed with flow cytometry between Days 1 and 15 to measure PLT-monocyte aggregate (PMA) and PLT-neutrophil aggregate (PNA) formation, WBC apoptosis (annexin V binding), and cell death (binding of 7-aminoactinomycin D). Procoagulant activity in the supernatant of four RBC preparations was assessed between Days 1 and 39 using a clotting assay with and without the addition of an inhibitory anti-tissue factor (TF) antibody, αTF-5. RESULTS: PLA formation was extensive and maximal on Day 3 of storage (PNA, 23 ± 13%; PMA, 93 ± 4%; n = 6). Apoptosis was progressive throughout storage, with 95 ± 4% of neutrophils and 73 ± 19% of monocytes binding annexin V on Day 15. Cell death became measurable after apoptosis. Procoagulant activity was observed in all RBCs but with varying temporal patterns. It was partially TF dependent and removed with high-speed centrifugation, suggestive of an association with microparticles. CONCLUSION: The activation of PLTs during the storage of RBCs induces PLA formation that precedes WBC apoptosis and death. Procoagulant activity, likely associated with microparticles derived from apoptotic WBCs, may contribute to adverse effects of stored, nonleukoreduced RBCs.

Whole Blood Thrombin Generation in Severely Injured Patients Requiring Massive Transfusion.

BACKGROUND: Despite the prevalence of hypocoagulability after injury, the majority of trauma patients paradoxically present with elevated thrombin generation (TG). Although several studies have examined plasma TG post injury, this has not been assessed in whole blood. We hypothesize that whole blood TG is lower in hypocoagulopathy, and TG effectively predicts massive transfusion (MT). STUDY DESIGN: Blood was collected from trauma activation patients at an urban Level I trauma center. Whole blood TG was performed with a prototype point-of-care device. Whole blood TG values in healthy volunteers were compared with trauma patients, and TG values were examined in trauma patients with shock and MT requirement. RESULTS: Overall, 118 patients were included. Compared with healthy volunteers, trauma patients overall presented with more robust TG; however, those arriving in shock (n = 23) had a depressed TG, with significantly lower peak thrombin (88.3 vs 133.0 nM; p = 0.01) and slower maximum rate of TG (27.4 vs 48.3 nM/min; p = 0.04). Patients who required MT (n = 26) had significantly decreased TG, with a longer lag time (median 4.8 vs 3.9 minutes, p = 0.04), decreased peak thrombin (median 71.4 vs 124.2 nM; p = 0.0003), and lower maximum rate of TG (median 15.8 vs 39.4 nM/min; p = 0.01). Area under the receiver operating characteristics (AUROC) analysis revealed lag time (AUROC 0.6), peak thrombin (AUROC 0.7), and maximum rate of TG (AUROC 0.7) predict early MT. CONCLUSIONS: These data challenge the prevailing bias that all trauma patients present with elevated TG and highlight that deficient thrombin contributes to the hypocoagulopathic phenotype of trauma-induced coagulopathy. In addition, whole blood TG predicts MT, suggesting point-of-care whole blood TG can be a useful tool for diagnostic and therapeutic strategies in trauma.