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Purpose Dietary guidelines recommend to limit egg consumption to 4 servings per week but the relation between egg intake and health outcomes is still controversial. To evaluate the association of egg consumption and mortality risk in Italian adults and to investigate nutritional factors and serum lipids as potentially explaining such associations. Methods Longitudinal analysis on 20,562 men and women aged ≥ 35y, free from cardiovascular disease (CVD) and cancer belonging to the Moli-sani Study cohort (enrolled 2005–2010) followed up for a median of 8.2 years. Results In multivariable-adjusted analysis as compared to low intake (> 0 ≤ 1 egg/week), eating > 4 eggs/week led to an increased risk of all-cause (Hazard ratio [HR] = 1.50; 95%CI 1.13–1.99), CVD (HR = 1.75; 1.07–2.87) and cancer mortality (HR = 1.52; 0.99–2.33). Similarly, an intake of 2–4 eggs/week was associated with higher all-cause (HR = 1.22; 1.01–1.46) and CVD mortality risk (HR = 1.43; 1.03–1.97). An increase of 1 egg per week was associated with higher mortality risk among high-risk individuals, such as those with hypertension and hyperlipidaemia. Dietary cholesterol explained about 43.0% and 39.3% (p values e.g., total cholesterol) accounted for a small proportion of egg-mortality relation. Conclusions Among Italian adults, high egg consumption leads to an increased risk of all-cause and CVD mortality, with the risk being evident even at the recommended intake of 2–4 eggs per week. A substantial part of this association was likely due to the egg contribution to dietary cholesterol. Our findings suggest limiting the consumption of eggs in the diet and these results should be considered in the development of dietary guidelines and updates.

Biological age (BA) is the hypothetical underlying age of an organism and has been proposed as a more powerful predictor of health than chronological age (CA). The difference between BA and CA (Δage) reflects the rate of biological aging, with lower values indicating slowed-down aging. We sought to compare the relationship of four a priori-defined dietary patterns, including a traditional Mediterranean diet (MD) and three non-Mediterranean diets, with biological aging (Δage) among Italian adults. We also examined distinctive nutritional traits of these diets as potential mediators of such associations.Cross-sectional analysis on a sub-cohort of 4510 subjects (aged ≥35 y; 52.0% women) from the Moli-sani Study (enrolment, 2005-2010). Food intake was recorded by a 188-item semi-quantitative food-frequency questionnaire. A Mediterranean diet score (MDS) was used as exposure and compared with non-Mediterranean dietary patterns, i.e. DASH (Dietary Approaches to Stop Hypertension), Palaeolithic and the Nordic diets. A Deep Neural Network based on 36 blood biomarkers was used to compute BA and the resulting Δage (BA-CA), which was tested as outcome in multivariable linear regressions adjusted for clinical factors, lifestyles and sociodemographic factors.In a multivariable-adjusted model, 1 standard deviation increase in the MDS was inversely associated with Δage (β = -0.23; 95%CI -0.40, -0.07), and similar findings were observed with the DASH diet (β = -0.17; 95%CI -0.33, -0.01). High dietary polyphenol content explained 29.8% (p = 0.04) and 65.8% (p = 0.02) of these associations, respectively, while other nutritional factors analysed (e.g. dietary fibre) were unlikely to be on the pathway. No significant associations were found with either the Palaeolithic or the Nordic diets.Increasing adherence to either the traditional MD or the DASH diet was associated with delayed biological aging, possibly through their high polyphenol content.

Objectives The aim of this study was to assess the hypothesis that alcohol consumption is associated with onset of atrial fibrillation (AF) and/or heart failure (HF). Background The connection between ethanol intake and AF or HF remains controversial. Methods The study population was 22,824 AF- or HF-free subjects (48% men, age ≥35 years) randomly recruited from the general population included in the Moli-sani study, for whom complete data on HF, AF, and alcohol consumption were available. The cohort was followed up to December 31, 2015, for a median of 8.2 years (183,912 person-years). Incident cases were identified through linkage to the Molise regional archive of hospital discharges. Hazard ratios were calculated using Cox proportional hazard models and cubic spline regression. Results A total of 943 incident cases of HF and 554 of AF were identified. In comparison with never drinkers, both former and occasional drinkers showed comparable risk for developing HF. Drinking alcohol in the range of 1 to 4 drinks/day was associated with a lower risk for HF, with a 22% maximum risk reduction at 20 g/day, independent of common confounders. In contrast, no association of alcohol consumption with onset of AF was observed. Very similar results were obtained after restriction of the analyses to regular or only wine drinkers or according to sex, age, social status, or adherence to the Mediterranean diet. Conclusions Consumption of alcohol in moderation was associated with a lower incidence of HF but not with development of AF.

Background Chili pepper is a usual part of a traditional Mediterranean diet. Yet epidemiological data on the association between chili pepper intake and mortality risk are scarce, with a lack of studies from Mediterranean populations. Objectives This study sought to examine the association between chili pepper consumption and risk of death in a large sample of the adult Italian general population, and to account for biological mediators of the association. Methods Longitudinal analysis was performed on 22,811 men and women enrolled in the Moli-sani Study cohort (2005 to 2010). Chili pepper intake was estimated by the EPIC (European Prospective Investigation Into Cancer) Food Frequency Questionnaire and categorized as none/rare consumption, up to 2 times/week, >2 to ≤4 times/week, and >4 times/week. Results Over a median follow-up of 8.2 years, a total of 1,236 deaths were ascertained. Multivariable hazard ratios (HRs) for all-cause and cardiovascular disease (CVD) mortality among participants in the regular (>4 times/week) relative to none/rare intake were 0.77 (95% confidence interval [CI]: 0.66 to 0.90) and 0.66 (95% CI: 0.50 to 0.86), respectively. Regular intake was also inversely associated with ischemic heart disease (HR: 0.56; 95% CI: 0.35 to 0.87) and cerebrovascular (HR: 0.39; 95% CI: 0.20 to 0.75) death risks. The association of chili pepper consumption with total mortality appeared to be stronger in hypertension-free individuals (p for interaction = 0.021). Among known biomarkers of CVD, only serum vitamin D marginally accounted for such associations. Conclusions In a large adult Mediterranean population, regular consumption of chili pepper is associated with a lower risk of total and CVD death independent of CVD risk factors or adherence to a Mediterranean diet. Known biomarkers of CVD risk only marginally mediate the association of chili pepper intake with mortality.

The effect of the polyphenol content of the human diet on mortality risk is not yet fully understood. The aim of this study was to evaluate the association of a polyphenol-rich diet with mortality rate and a possible mediation effect by inflammation, in what we believe to be a novel, holistic approach.We analyzed 21 302 participants (10 980 women and 10 322 men, aged ≥35 y) from the Moli-sani cohort. The participants were followed up for a median of 8.3 y. The European Prospective Investigation into Cancer and Nutrition food frequency questionnaire (FFQ) was used for dietary assessment. Flavonol, flavone, flavanone, flavanol, anthocyanin, isoflavone, and lignan intakes were calculated using European Food Information Resource-Bioactive Substances in Food Information Systems and the polyphenol antioxidant content (PAC)-score was constructed to assess the total content of these nutrients in the diet.Participants included in the highest quintile of intake of various polyphenol classes and subclasses presented a significant lower all-cause mortality risk compared with those in the lowest group of consumption (hazard ratio [HR] 1; P 0.05). Cox regression analyses adjusted for potential confounders indicated that participants in higher quintiles of PAC-score had lower all-cause mortality risk (HR1; P 0.05). When cause-specific mortality rates were considered, similar effects were observed for cardiocerebrovascular and cancer mortality (HR1; P 0.05).The polyphenol content of the diet was associated with reduced mortality risk in a Mediterranean population, possibly through an antiinflammatory mechanism.

Evidence indicates that Vitamin D deficiency may be associated with increased risk of cardiovascular disease, although findings on risk of heart failure (HF) are controversial. We investigated the relationship between serum Vitamin D and the incidence of hospitalization for HF in a large prospective cohort of Italian adults.19,092 (49% men, age range 35-99 years) HF-free individuals from the Moli-sani study, with complete data on serum Vitamin D (25-hydroxyvitamin) levels and incident hospitalized HF, were analysed. The cohort was followed up for a median of 6.2 years. Baseline serum Vitamin D levels were categorized in deficient (10 ng/mL), insufficient (10-29 ng/mL), and normal (≥30 ng/mL) Incident cases of hospitalization for HF were identified by linkage with the regional hospital discharge registry. Hazard ratios (HRs) were calculated using Cox-proportional hazard models. The prevalence of normal, insufficient or deficient levels of Vitamin D was 12.2%, 79.6% and 8.2%, respectively. During follow-up, 562 admissions to hospital for HF were identified. The incidence of HF was 1.6%, 2.9% and 5.3%, respectively in subjects with normal, insufficient and deficient levels of Vitamin D. After multivariable analysis, individuals with deficiency of Vitamin D had a higher risk of hospitalization for HF (HR: 1.61, 95%CI: 1.06-2.43) than those with normal levels. Further adjustment for subclinical inflammation did not substantially change the association between Vitamin D deficiency and HF.Deficiency of Vitamin D was associated, independently of known HF risk factors, with an increased risk of hospitalization for HF in an Italian adult population.

Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 ± 377 seconds) and arterioles (858 ± 289 seconds) than in WT vessels (559 ± 241 seconds, P < .001; 443 ± 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.

Background— Particulate air pollution is associated with cardiovascular diseases and myocardial infarction (MI). Methods and Results— We investigated the relationship between airway inflammation and thrombosis 24 hours after intratracheal (IT) instillation of diesel exhaust particles (DEP; 50 μg/hamster). Mild thrombosis was induced in the femoral vein by endothelial injury, and the consequences of airway inflammation on thrombogenicity were studied via online video microscopy. Lung inflammation and histamine analysis in bronchoalveolar lavage (BAL) and plasma were performed after pretreatment with dexamethasone (DEX) or sodium cromoglycate (SC). DEP induced airway inflammation and histamine release in BAL and in plasma, and increased thrombosis, without elevating plasma von Willebrand factor (vWF) levels. The IT instillation of 400-nm positively charged polystyrene particles (500 μg/hamster), serving as particles that do not penetrate into the circulation, equally produced airway inflammation, histamine release, and enhanced thrombosis. Histamine in plasma resulted from basophil activation. Intraperitoneal (IP) pretreatment with DEX (5 mg/kg) abolished the DEP-induced histamine increase in BAL and plasma and abrogated airway inflammation and thrombogenicity. The IT pretreatment with DEX (0.5 mg/kg) showed a partial but parallel inhibition of all of these parameters. Pretreatment with SC (40 mg/kg, IP) strongly inhibited airway inflammation, thrombogenicity, and histamine release. Conclusions— Our results are compatible with the triggering of mast cell degranulation and histamine release by DEP. Histamine plays an initial central role in airway inflammation, further release of histamine by circulating basophils, and peripheral thrombotic events. Antiinflammatory pretreatment can abrogate the peripheral thrombogenicity by preventing histamine release from mast cells.

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.

Although drugs exist for the primary and secondary prevention of thrombosis, more potent antiplatelet drugs with sufficiently wide therapeutic windows to avoid bleeding complications are needed. Both academic and pharmaceutical laboratories are working to develop such drugs. This chapter reviews the potential of inhibiting interactions between von Willebrand factor (vWF) and the second most abundant receptor on the platelet, the glycoprotein (GP) Ib/IX/V complex, interactions that are essential for the activation of circulating platelets, contacting a vessel wall injury. Although still at the level of preclinical testing, this area is expected to progress quickly during the next few years, also in view of the three-dimensional structural information that has recently become available and that allows a molecular understanding of vWF binding to the GPIbalpha chain of the GPIb complex.

We have generated transgenic mice overexpressing the human P2X1 ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X1ionotropic activity as determined by more prominent P2X1-mediated Ca2+ influx and platelet shape change. P2X1 overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A2 mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds−1 resulted in increased P2X1-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X1 ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A2-, and shear stress–triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbalpha. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbalpha-binding site. Glutathione-S-transferase (GST)-human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin- or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbalpha binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIbalpha-binding site, located around the front pocket of the A1 domain and defined by strands beta3, beta4, and helix alpha3, and it provides a mechanistic basis for VWF neutralization by this antibody.

SummaryAlternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein α-subunit (Gsα) and the extra-large stimulatory G-protein α-subunit (XLsα), are located on the imprinted chromosomal region 20q13.12-13. We presently report a functional polymorphism in the imprinted XL-GNAS1 gene. In three patients, a 36 bp insertion and two basepair substitutions flanking this insertion were found in the paternally inherited XL-GNAS1 exon 1. They clinically manifest an enhanced trauma-related bleeding tendency and a variable degree of mental retardation. A platelet aggregation inhibition test to evaluate Gs function was developed. Their platelets display Gs hyperfunction and an enhanced cAMP generation upon stimulation of Gs-coupled receptors. The prevalence of the XLsα insertion in a normal control group was 2.2%. Normal controls, inheriting the insertion maternally, had a normal platelet Gs activity, whereas controls inheriting the insertion paternally had increased inducible platelet Gs activity, defining the insertion as a functional polymorphism. This paternally inherited XLsα insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.

SummaryATP is a potent agonist of the P2X1 ion channel, mediating a rapid, quickly desensitized influx of Ca2+. In hirudinized PRP, containing apyrase, the two stable selective P2X1 agonists, α,β-methylene ATP, and L-β, γ-methylene ATP induced extracellular Ca2+-dependent fast and reversible platelet shape change, leading to desensitization of the P2X1 ion channel. Preincubation with HPLC-purified ADP potently antagonized the subsequent α, β-methylene ATP- and L-β, γ-methylene ATP-evoked platelet shape change. Accordingly, upon heterologous expression of P2X1 in Xenopus oocytes, HPLC-purified ADP acted as an antagonist of the ATP-induced current, but was inactive itself. Since ATP and ADP are co-released from dense granules during platelet activation, we investigated whether the P2X1 ion channel is involved in the response of platelets to collagen. We found that platelet shape change and aggregation induced by low concentrations of collagen were strongly inhibited after selective desensitization of P2X1 with its agonists or by pretreating the platelets with a low concentration of ADP (0.5 μM), that antagonizes the P2X1 channel without desensitizing the P2Y1 receptor. Our data suggest that, during collagen-initiated platelet activation, the early secretion of ATP results in the activation of the P2X1 ion channel, which plays a role as a positive regulator of further platelet responses.

Background/Methods: Anti-von Willebrand factor (vWf) antibody mediated platelet activation was studied using 2 monoclonal anti-vWf antibodies promoting the binding of vWf to GPIbα: 1C1E7 (IgG2a) reacting with the vWf N-terminus and 75H4B12 (IgM), characterized in this paper and studied in association with 1C1E7. Results: 75H4B12 binds to an N-terminal epitope in vWf, different from that reacting with 1C1E7. When com-bined, 1C1E7 and 75H4B12 promoted vWf binding to isolated GPIb under static conditions, even in the absence of ristocetin or botrocetin, and induced platelet aggregation synergistically in the presence of zero to subthreshold ristocetin concentrations. Specific inhibitors of GPIbα-vWf interactions prevented vWf binding to GPIbα in ELISA and during platelet aggregation. In addition, the 1C1E7 dependent platelet aggregation involved Fc receptor mediated platelet activation, a phenomenon even more pronounced when 1C1E7 and 75H4B12 were combined. A 75H4B12 binding phage expressing a peptide homologous with vWf sequence 88–95 neutralized the antibody induced platelet activation. However, at arterial shear rates, both 1C1E7 and 75H4B12 potently prolonged cartridge closure times in the PFA-100, compatible with inhibition of platelets by vWf, unfolded by the combined action of shear stress and antibodies. Conclusions: We conclude that antibodies directed against different epitopes in the N-terminus of vWf modify the folded vWf structure synergistically and enhance A1 domain mediated vWf binding to platelet GPIb at low shear forces. In addition, once platelet-bound, IgG antibodies potently activate platelets via the FcγII receptor. Thus, such antibodies may promote immune mediated thrombosis at low shear rates, typical of the venous circulation. In contrast, at arterial shear rates, anti-vWf antibodies may rather compromise platelet function following enhanced binding of the unfolded vWf multimers to platelets, shielding platelets from interacting with subendothelial and soluble ligands.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A. ispartof: Blood vol:96 issue:3 pages:958-65 ispartof: location:United States status: published

The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 +/- 19 x 10(6) arbitrary light units [AU] n = 15, mean +/- SEM), thrombosis in PAI-1(-/-) mice (40 +/- 10 x 10(6) AU, n = 13) was inhibited (P

SummaryA Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 X 106 cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor’s antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.

Circulating complexes of leukocytes and activated platelets are markers for atherosclerosis, but their interaction with the arterial endothelial lining has not been studied. Therefore, the effect of activated platelets on rolling and adhesion of labeled human THP-1 monocytoid cells to human umbilical vein endothelial cell (HUVEC) monolayers was studied by epifluorescence microscopy in a parallel plate flow chamber. In the absence of activated platelets, THP-1 rolling on resting HUVEC was negligible at shear rates greater than 300 s(-1). Activation of HUVEC with 100 nmol/L phorbol myristate acetate (PMA) increased THP-1 cell adhesion at shear rates less than 400 s(-1). Therefore, a shear rate of 400 s(-1) was identified as a threshold for THP-1 adhesion. THP-1 rolling on activated HUVEC was reduced by 64% after L-selectin inhibition but was not affected by P-selectin inhibition. The addition of 1 to 50 thrombin receptor-activating peptide (TRAP)-activated platelets per THP-1 cell enhanced interactions between THP-1 cells and HUVEC, resulting in a steep bell-shaped dose-response curve, with a peak of 10 +/- 3 rolling cells/50 seconds at 3 platelets per THP-1 cell (P

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

SummaryThe thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 g/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.

SummaryA mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wild-type from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4κ monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass ∼7.4 × 105M−1 s−1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss ≤1 × 10−5s−1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4κ monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass ∼7.4 × 105M−1 s−1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss ≤1 × 10−5s−1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

A patient with a history of recurrent late fetal loss associated with multiple placental infarcts and cerebrovascular ischemia at the age of 36, followed a year later by a myocardial infarction, was referred for further investigation. Coronary angiography was normal. Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies, and other thrombophilia parameters were negative, but there was moderate hyperthyroidism with positive thyroid peroxidase antibodies. Platelet numbers and von Willebrand factor (vWF) were normal. Her platelets showed spontaneous aggregation that disappeared with aspirin intake. However, aggregation still was induced by low levels of ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in patient platelet-rich plasma (PRP) was completely blocked by neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The monoclonal anti-FcγRII receptor antibody IV.3 inhibited partly, which suggests that PRP aggregation by low-dose ristocetin was elicited by vWF-immunoglobulin (Ig) complexes. Upon addition to washed human platelets, with vWF (10 μg/mL), purified patient Igs dose-dependently enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 μg/mL, an effect that disappeared again above 1 mg/mL. Aggregation was dependent on the vWF concentration and was blocked by IV.3 or neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous aggregation of normal platelets resuspended in patient plasma could be inhibited totally by IV.3 and partially by neutralizing anti-GPIb or anti-vWF antibodies. Perfusion with normal anticoagulated blood, enriched with 10% of control or patient plasma, over surfaces coated with vWF showed increased platelet adhesion and activation in the presence of patient antibodies. Treatment of the patient with the antithyroid drug thiamazol and temporary corticosteroids, aspirin, and ticlopidine did not correct the platelet hypersensitivity to ristocetin. These observations suggest that some autoantibodies to vWF may both enhance vWF binding to platelets and cause platelet activation through binding to the FcγRII receptor, and thereby may be responsible for a new form of antibody-mediated thrombosis.

Summaryvon Willebrand factor (vWF) is instrumental in arterial but has also been implicated in venous thrombogenesis. To address its role in venous thrombosis, experimental thrombosis was induced in the carotid artery and the femoral vein of hamsters, following which thrombus prevention by two different antagonists of vWF was studied. The first antagonist was the anti-human vWF monoclonal antibody AJvW-2, which inhibits the botrocetin and ristocetin induced aggregation of human blood platelets. AJvW-2 reacts with an epitope present in the A1 domain of vWF in very different species (human, pig, rabbit, dog, Guinea pig and rat). This epitope was found to be conformational and overlapping with vWF binding sites for aurin tricarboxylic acid (ATA), but not for botrocetin and heparin. AJvW-2 has affinities for vWF in the absence (Kd = 0.5 ± 0.03 nmol/l in solution) and in the presence of shear stress (Kd = 3.3 ± 0.6 nmol/l during perfusion at 1,300 s–1 over subendothelial matrix associated vWF) sufficiently elevated to neutralize vWF. During perfusion of subendothelial matrix with anticoagulated human blood, the surface covered by adhering platelets was reduced by AJvW-2, with IC50s equal to 6.6 ± 0.34 μg/ml at 1,300 s–1 and to 1 ± 0.01 μg/ml at 2,700 s-1. As a second antagonist, molecular size gel filtered ATA was selected. Fractionated ATA inhibited platelet adhesion to matrix with IC50s equal to 0.27 ± 0.09 mmol/l at 1,300 s –1 and 0.16 ± 0.008 mmol/l at 2,700 s –1. When administered to hamsters, AJvW-2 prevented thrombosis in the injured carotid artery dose-dependently (ED50 = 0.15 ± 0.01 mg/kg). Thrombosis in the similarly injured femoral vein was however also inhibited (ED50 = 0.37 ± 0.06 mg/kg). Likewise, fractionated ATA completely inhibited carotid artery thrombosis (ED50 = 0.42 ± 0.13 mg/kg), but also interfered with femoral vein thrombosis (apparent ED50 between 2 and 3 mg/kg). We conclude that antagonizing the vWF A1 domain by AJvW-2 and to a lesser extent also by fractionated ATA, inhibits thrombosis not only in the arterial but also in the venous circulation. Since venous thrombi were prevented at only 3-5-fold higher doses of antagonist, vWF participates in injury induced venous thrombosis.

SummaryWe recently described an outbreak of anti-factor VIII (FVIII) antibodies in a population of haemophilia A patients non-responsive to FVIII (1). To find out what part of the FVIII molecule had been altered, we purified specific anti-FVIII antibodies from the plasma of the five patients showing high titres of inhibitors. An average of 100 µg antibodies per ml of initial plasma was recovered by immunoadsorption on insolubilised FVIII. The antibodies followed the normal isotypic distribution, including the presence of specific IgG2 antibodies; the relative increase in IgG4 that is usually observed in patients with long-standing inhibitors, was not present. The regions of FVIII to which human antibodies bound were determined by a competition assay using a panel of murine monoclonal antibodies: two major regions were identified, one located in the A2 heavy chain domain, and the other made of determinants of both the A3 and C2 light chain domains. Affinity-purified antibodies inhibited the function of FVIII as determined in a chromo- genic assay. However, variations existed in the affinities with which antibodies bound to soluble FVIII. This study shows that the immu- nogenicity of two particular regions of FVIII has been altered. A screening for alterations located in these two regions should possibly be included in the preclinical evaluation of FVIII concentrates.

After a standardized trauma to carotid arteries or femoral veins of hamsters, the antithrombotic effects of two antiplatelet agents (aspirin and the glycoprotein llb/llla antagonist G4120) and two anticoagulants (heparin and the direct thrombin inhibitor rhirudin) were studied in vivo. The thrombus area volume was assessed by image analysis of the transilluminated experimental vessels. Heparin, r-hirudin, and G-4120 demonstrated a dosedependent complete inhibition of arterial and venous thrombosis. In contrast, the antithrombotic effect of aspirin was only partial in both vessel types. A significant correlation between activated partial thromboplastin time (aPTT) at the end of the experiments and the antithrombotic effect was observed with the anticoagulant agents. However, only r-hirudin inhibited thrombus formation at a therapeutical prolongation of aPTT, while heparin required supratherapeutical amounts to achieve the same inhibition. The data confirm that the inhibition of aspirin, heparin, r-hirudin, and G-4120 on the formation of platelet-rich thrombi is independent of the blood flow rate. Stockmans F, Stassen JM, Vermylen J, Hoylaerts MF, Nystrom A. A technique to investigate microvascular mural thrombus formation in arteries and veins. II. Effects of aspirin, heparin, r-hirudin, and G-4120. Ann Plast Surg 1997;38:63-68

SummaryPrevious investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2,7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor Vlla-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.Thus the investigated aprotinin-derived coagulation inhibitors resulted in a series of combined coagulation inhibitors with a pharmacological behaviour, which justifies in vivo testing of their potential antithrombotic action, as reported in the accompanying paper.

MK-383 (L-tyrosine, N-n-butylsulfonyl)-O-[4-butyl(4-piperidinyl)], monohydrochloride monohydrate) is a potent and specific platelet fibrinogen receptor antagonist that may be useful in preventing processes that lead to occlusive thrombus formation in the lumen of the blood vessel. Two placebo-controlled phase I trials were completed in 56 healthy volunteers to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of MK-383 administered as 1- and 4-hour infusions in the presence and absence of aspirin. When administered to healthy male subjects by constant infusions up to 0.4 µg/kg/min over 1 hour or up to 0.2 µg/min over 4 hours, it provided a well-tolerated reversible means of inhibiting platelet function. At infusion rates of 0.25 and 0.15 µg/kg/min for 1 and 4 hours, respectively, MK-383 extended baseline bleeding time by 2.0- to 2.5-fold and inhibited adenosine diphosphate (ADP)-induced platelet aggregation by at least 80%. The pharmacokinetics of MK-383 include a mean plasma clearance of 329 ml/min, steady-state volume of distribution of 76 L, and half-life of 1.6 hours. The percentage of dose excreted in the urine was 37%. Correlations between MK-383 plasma concentration (C) and inhibition of platelet aggregation were examined by fitting with a sigmoid maximum-effect model. The plasma concentration yielding 50% inhibition (C50) for MK-383 in healthy volunteers is approximately 13 ng/ml, with a Hill coefficient >5. Based on a naive pooled analysis, an exponential empirical model best describes the MK-383 C–extension of template bleeding time (BTE) relationship. The model indicates that the MK-383 plasma concentration necessary to double BTE is approximately 30 ng/ml (i.e., 2.5-fold greater than the C50 for ADP-induced inhibition of platelet aggregation). The pharmacokinetics of MK-383 was unaffected by pretreatment with 325 mg aspirin 1 day before and 1 hour before infusion. Conversely, aspirin pretreatment reduced C50 and increased bleeding time extension, suggesting that aspirin may have an additive effect with respect to inhibition of platelet function. Based on the putative role of the fibrinogen receptor in thrombotic processes and an acceptable human pharmacokinetic-pharmacodynamic profile, MK-383 should be evaluated in patients with unstable angina. Clinical Pharmacology and Therapeutics (1994) 56, 377–388; doi:10.1038/clpt.1994.152

Ten healthy male volunteers not previously exposed to hirudin participated in an open trial to evaluate the effects on platelet function and coagulation of recombinant hirudin (rec-hirudin) CGP 39393 1 mg/kg administered intravenously as a bolus injection.

In a recent issue of Blood, Greco et al[1][1]reported on the expression of a novel structurally altered P2X1 receptor in platelets and in megakaryocytic cell lines. This P2X1 variant lacks 17 amino acids in its extracellular domain due to a deletion within exon 6 of the P2X1 gene (GenBank accession

A significant proportion of hemophilia A patients receiving transfusions of factor VIII (FVIII) develop a specific antibody response towards FVIII. These antibodies are usually detected by assays in which they inhibit the function of the molecule, such as the Bethesda clotting test. We have prepared anti-FVIII antibodies by specific immunoadsorption from the plasma of four hemophiliacs with stable inhibitor levels. The isotypic distribution of such antibodies was determined and their capacity to bind to insolubilized FVIII was compared with their inhibitory activity in two functional assays, namely, the Bethesda assay and a chromogenic assay. In addition, the FVIII epitope specificity was determined by competition with monoclonal antibodies for the binding to insolubilized FVIII. We show here that (1) anti-FVIII antibodies are not isotypically restricted; thus, a significant proportion of specific IgG2 was found; (2) antibodies are frequently directed towards epitopes of FVIII that are not directly involved in the function of the molecule and therefore escape detection in the Bethesda method or chromogenic assay; and (3) each patient shows a unique pattern of FVIII epitope recognition. We conclude that evaluation of anti-FVIII antibodies by a functional method does not provide an accurate evaluation of the specific antibody response. These findings have important implications for the comparison of the immunogenicity of FVIII molecules produced by different technologies and for the development of methods to control anti-FVIII antibody production.

We here present an easily standardizable and reproducible procedure which clearly separates lupus anticoagulants (LA) from coagulation factor inhibitors. This new LA neutralization test makes use of platelet-derived microvesicles which were prepared as follows: gel-filtered platelets (4 x 10(5)/microliters) were incubated with 60 microM of the calcium ionophore A23187 for 20 min at 37 degrees C. The vesicles were separated from the platelet aggregates by centrifugation at 1000 g for 10 min. The vesicle containing supernatant was then spun down at 15,000 g for 15 min, lyophilized and stored at -20 degrees C until used. The vesicles were resuspended in plasma from normal individuals, from patients with LA activity, from patients with factor VIII inhibitors, from patients with congenital factor deficiencies and from patients receiving oral anticoagulants or intravenous heparin. A kaolin clotting time was performed in the absence (KCT) or presence of these vesicles (KCTves) and the ratios of these times to their respective mean normal times were calculated. Segregation of LA patients from all remaining patients except heparinized ones could be made with a high degree of accuracy. A thrombin time was needed to separate LA from heparinized patients. The method was highly reproducible and only minor (negligible) differences in potencies were observed between different vesicle preparations. Both the intra-batch and the inter-batch coefficients of variations on the KCTves were lower than 6%.

Summary. We describe a patient with a lifelong bleeding disorder previously classified as von Willebrand's disease (vWD) type I. The factor VIII (FVIII) level in this patient was disproportionately low and we showed that this was due to a decreased factor VIII binding capacity of her vWF. To characterize the molecular defect in this type of vWD, a cDNA-dependent polymerase chain reaction (PCR) amplification was performed using platelet RNA as a template. Direct sequencing of the amplified fragment, which encodes for the FVIII-binding domain, showed a single nucleotide change in exon 20 at codon 854, resulting in the substitution of CAG glutamine (Gln) for CGG arginine (Arg). At the level of the cDNA only the mutated sequence was found, whereas at genomic DNA level the patient was heterozygous for this mutation. This patient is therefore a compound heterozygote for a point mutation resulting in a FVIII-binding defect and a vWF allele with low transcript levels.

ADP plays a central role in platelet aggregation. Activation of platelets via ADP proceeds via three membrane receptor proteins, two of which are coupled to a G protein and the third one constituting an ion channel mediating rapid Ca2+-influx. Via Ca2+-mobilization, the Gq-coupled P2Y1 receptor acts in concert with the Gi-coupled P2TAC receptor, which functions by lowering intracellular cAMP levels. The importance of Ca2+-influx via the P2X1 ion channel remains to be elucidated.

SummaryIt was the aim of the present study to perform a systematic review of the published studies that estimated the prevalence of non-responders to aspirin, as assessed by the closure time of PFA-100®, a point-of-care device, and to analyse: 1) some major clinical and methodological factors that can influence it and 2) its possible association with vascular outcomes. The prevalence of non-responders to aspirin in 64 populations from 53 studies, comprising 6,450 subjects, had a median value of 0.27.A higher number of aspirin non-responders was found among older patients, those with acute vascular events, or those treated for more than one month. Aspirin non-response was more frequently associated with the use of “home-established” cut-offs or when closure time was only assessed after aspirin (rather than both before and after).Among risk factors, type 2 diabetes appeared to be associated with a higher prevalence of aspirin non-responders. The latter was also higher in less recent publications and in studies that used 3.2% rather than 3.8% Na-citrate as an anticoagulant. In eight studies comprising 847 subjects, aspirin non-responders were more likely to have vascular events than responders (relative risk: 1.63; 95% CI 1.16–2.28). In conclusion, although there appears to be heterogeneity among the studies analysed, this review indicates that about one quarter of people receiving aspirin would be identified – as an average – as aspirin non-responders by PFA-100. As this is a simple, widely available point-of-care test, efforts to better standardize it and to control for its major methodological variables might be useful to improve monitoring of platelet performance under aspirin treatment and to firmly establish the observed association with clinical vascular events.

Preface 1. The structure and production of blood platelets Joseph E. Italiano, Jr 2. Platelet immunology: structure, functions and polymorphisms of membrane glycoproteins Yasuo Ikeda, Yumiko Matsubara and Tetsuji Kamata 3. Mechanisms of platelet activation Lawrence Brass and Timothy J. Stalker 4. Platelet priming Paolo Gresele, Emanuela Dalcinelli and Stafania Momi 5. Platelets and coagulation Jose A. Lopez and Ian del Conde 6. Vessel-wall-derived substances affecting platelets Azad Raiesdana and Joseph Loscalzo 7. Platelet-Leukocyte-Endothelial cross talk Kevin J. Croce, Masashi Sakuma and Daniel I. Simon 8. Laboratory investigation of platelets Eduard Shantsila, Timothy Watson and Gregory Y. H. Lip 9. Clinical approach to the bleeding patient Jos Vermylen and Kathelijne Peerlinck 10. Thrombocytopenia James Bussel and Andrea Primiani 11. Reactive and clonal thrombocytosis Ayalew Tefferi 12. Congenital disorders of platelet function Marco Cattaneo 13. Acquired disorders of platelet function Michael H. Kroll and Amy A. Hassan 14. Platelet transfusion therapy Sherrill J. Slichter and Ronald G. Strauss 15. Clinical approach to the patient with thrombosis Brian G. Choi and Valentin Fuster 16. Pathophysiology of arterial thrombosis Juan Jose Badimon, Borja Ibanez and Gemma Vilahur 17. Platelets and atherosclerosis Stephan Lindemann and Meinrad Gawaz 18. Platelets in other thrombotic conditions David L. Green, Peter W. Marks and Simon Karpatkin 19. Platelets in respiratory disorders and inflammatory conditions Paolo Gresele, Stefania Momi, Simon C. Pitchford and Clive P. Page 20. Platelet pharmacology Dermot Cox 21. Antiplatelet therapy versus other antithrombotic strategies Nicolai Mejevoi, Catalin Boiangiu and Marc Cohen 22. Laboratory monitoring of antiplatelet therapy Paul Harrison and David Keeling 23. Antiplatelet therapy in cardiology Pierluigi Tricoc and Robert A. Harrington 24. Antiplatelet therapy in cerebrovascular disease James Castle and Gregory W. Albers 25. Antiplatelet treatment in peripheral arterial disease Raymond Verhaeghe and Peter Verhamme 26. Antiplatelet treatment of venous thromboembolism Menno V. Huisman, Jaapjan D. Snoep, Jouke T. Tamsma and Marcel M. C. Hovens Index.

SummaryPlatelets from a patient with an acquired hemorrhagic disorder had a severely impaired response to collagen, whereas platelet aggregation to other agonists and coagulations tests were normal. No abnormalities of the patient’s platelet membrane glycoproteins (GP) were seen.Tleatment of the patient with immunosuppressive agents temporarily improved both the bleeding tendency and the collagen responsiveness of the platelets. An IgG was found to be present in the plasma, directed against a protein comigrating with GPIa, and coadsorbing with GPIa to insoluble collagen fibers in a Mg+2-dependent manner. Furtherrnore, GPIa was recognized by the patient’s antibody when affinity-purified GPIa-IIa was used as antigen. Finally, the GPIa-IIa complex was immunoprecipitated from a platelet lysate by patient’s plasma.In addition, purified platelet specific IgG’s from the patient inhibited aggregation of normal platelets induced by collagen or by wheat germ agglutinin. We conclude that the lack of response to collagen of the patient’s platelets may well be due to the presence of an autoantibody against GPIa.

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase–activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34+ cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34+ cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Short-term increases in particulate air pollution are associated with increased incidence of cardiovascular events. Previously, we showed that intratracheally instilled diesel exhaust particles (DEPs) are prothrombotic. Here, we investigated the time course and the mechanisms. At 1, 6, and 24 hours after instillation of 50 microg DEPs per hamster, the mean size of in vivo-induced and quantified venous thrombosis was increased by 480%, 770%, and 460%, respectively. Platelets activation in blood was confirmed by a shortened closure time in the platelet function analyzer (PFA-100). In bronchoalveolar lavage, neutrophils and histamine levels were increased at all time points. In plasma, histamine was increased at 6 and 24 hours but not at 1 and 3 hours. Pretreatment with a histamine H1-receptor antagonist (diphenhydramine, 30 mg/kg intraperitoneally) abolished the DEP-induced neutrophil influx in bronchoalveolar lavage at all time points. However, diphenhydramine pretreatment did not affect DEP-induced thrombosis or platelet activation at 1 hour, whereas both were markedly reduced at 6 and 24 hours. In conclusion, pulmonary inflammation and peripheral thrombosis are correlated at 6 and 24 hours, but at 1 hour, the prothrombotic effects do not appear to result from pulmonary inflammation but possibly from the blood penetration of DEP-associated components or by DEP particles themselves.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients' platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively. ispartof: Journal of Clinical Investigation vol:113 issue:6 pages:905-912 ispartof: location:United States status: published