Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases.

Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d-/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2-/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2-/- and Unc13d-/- mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.

Functional Classification of Paediatric Patients with Non-syndromic Delta-Storage Pool Deficiency.

Storage pool disease (SPD) covers a group of platelet defects in which α- and/or delta-granules are reduced or cannot be secreted adequately in response to agonists. The detection of delta-granule release defects is hampered by a lack of fast and feasible tests. We aimed to implement a flow cytometry-based kinetic mepacrine assay to better identify and subgroup childhood patients with a mild to moderate bleeding diathesis and compare our method to established laboratory tests. We analysed 50 children with suspected SPD whose initial parameters were re-assessed in a second site visit. Mepacrine uptake and release patterns were correlated with CD63 exposure, platelet ADP/ATP release and content, and the bleeding score ascertained by the ISTH-BAT. Mepacrine release was overall significantly reduced in investigated patients compared with controls. Summarizing, our time-resolved approach proved to be a quick and inexpensive tool that was additionally able to distinguish between mepacrine uptake, mepacrine release, and combined defects. Classification of patients using such a kinetic assay makes it feasible to sensitively detect frequently missed SPD and to group these patients for further analyses and clinical correlations.

[New oral anticoagulants for prophylaxis of stroke. Results of an expert conference on practical use in geriatric patients]. / Neue orale Antikoagulanzien zur Prophylaxe von Schlaganfällen. Ergebnis einer Expertenkonferenz zum praktischen Einsatz bei geriatrischen Patienten.

Geriatric patients with non-valvular atrial fibrillation (AF) are increasingly being treated with novel oral anticoagulants (NOAC) to prevent ischemic stroke. This article highlights the outcome of an expert meeting on the practical use of NOAC in elderly patients. An interdisciplinary group of experts discussed the current situation of stroke prevention in geriatric patients and its practical management in daily clinical practice. The topic was examined through focused impulse presentations and critical analyses as the basis for the expert consensus. The key issues are summarized in this paper. The European Society of Cardiology (ESC) guidelines from 2012 for the management of patients with non-valvular AF recommend NOAC as the preferred treatment and vitamin K antagonists (VKA) only as an alternative option. Currently, the NOAC factor Xa inhibitors apixaban and rivaroxaban and the thrombin inhibitor dabigatran are more commonly used in clinical practice for patients with AF. Although these drugs have many similarities and are often grouped together it is important to recognize that the pharmacology and dose regimes differ between compounds. Especially n elderly patients NOAC drugs have some advantages compared to VKA, e.g. less drug-drug interactions with concomitant medication and a more favorable risk-benefit ratio mostly driven by the reduction of bleeding. Treatment of anticoagulation in geriatric patients requires weighing the serious risk of stroke against an equally high risk of major bleeding and pharmacoeconomic considerations. Geriatric patients in particular have the greatest benefit from NOAC, which can also be administered in cases of reduced renal function. Regular control of the indications is indispensable, as also for all other medications of the patient. The use of NOAC should certainly not be withheld from geriatric patients who have a clear need for oral anticoagulation.

Cytomegalovirus infection is associated with venous thromboembolism of immunocompetent adults--a case-control study.

Cytomegalovirus (CMV) seems to contribute to the development of venous thromboembolism (VTE) in immunocompromised patients whereas literature data on the role in immunocompetent individuals are mainly limited to case reports. This study aimed to investigate if cytomegalovirus infection contributes to the development of VTE in immunocompetent individuals. CMV-IgG and CMV-IgM antibody titres, CMV-IgG avidity and CMV-DNA were identified in samples from 166 VTE patients and from 166 healthy blood donors matched for gender and age. CMV-IgG antibodies were found more frequently in VTE patients compared to controls [57.8% vs. 44.0%; adjusted OR 1.75 (95% CI 1.13-2.70); p = 0.016]. Accordingly, median CMV-IgG titres were significantly higher in the case group (89.4 vs. 1.8 AU/ml; p = 0.002). Although the overall rate was low, CMV-IgM antibodies were detected more often among cases than controls. The difference was significant in patients with an unprovoked VTE event [7.4% vs. 0.6%; adjusted OR 5.26 (95% CI 1.35-20.8); p = 0.017]. CMV-IgG antibodies of almost all VTE patients (98.9%) and controls (98.6%) were found to be of high avidity. The rate of positive CMV-DNA samples was low and not different between cases and controls. With the exception of age, no association was found between CMV seropositivity and established VTE risk factors within the VTE group. CMV infection seems to play a role in the development of VTE in immunocompetent patients. Recurrent infection might be more important than acute CMV infection.

Antibodies to platelet factor 4-heparin complex and outcome in hemodialysis patients with diabetes.

BACKGROUND AND OBJECTIVES: Hemodialysis patients with type 2 diabetes exhibit an excessive cardiovascular risk and regularly receive heparin. We tested whether antibodies to the platelet factor 4-heparin complex (PF4-H-AB) contribute to outcome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In 1255 hemodialysis patients with type 2 diabetes, the German Diabetes Dialysis Study evaluated the effect of atorvastatin (20 mg/d) versus placebo. In a post hoc analysis, the association among PF4-H-ABs, biochemistry, and prespecified, centrally adjudicated end points (combined cardiovascular end point [CVE], all-cause mortality, sudden death, myocardial infarction, stroke) was investigated. RESULTS: During 4 years, 460 patients reached the CVE; 605 died, 159 of sudden death. Myocardial infarction and stroke occurred in 199 and 97 patients, respectively. Positive PF4-H-AB status was found in 231 (18.7%) of 1236 tested patients and was associated with lower albumin, higher C-reactive protein, and arrhythmia. In a multivariate model adjusted for demographics, comorbidities, and biochemistry, PF4-H-ABs were associated with sudden death. No significant association between PF4-H-ABs and all-cause mortality, myocardial infarction, stroke, or the CVE was observed. Detecting an interaction between acetylsalicylic acid and PF4-H-ABs regarding sudden death and mortality, we found that the association between PF4-H-ABs and outcomes was restricted to patients with acetylsalicylic acid use, most likely because of indication bias. CONCLUSIONS: In hemodialysis patients who have type 2 diabetes and are treated with acetylsalicylic acid, PF4-H-ABs are associated with sudden and all-cause death. Further studies are needed to elucidate this association.

Association of ADAMDEC1 haplotype with high factor VIII levels in venous thromboembolism.

A suggestive locus on chromosome 8 could be shown to be associated with familial high factor VIII (FVIII) levels in venous thromboembolism. The ADAMDEC 1 gene is a candidate expressing an ectodomain sheddase. However, the ectodomain of the clearance receptor for FVIII, the low-density lipoprotein receptor-related protein (LRP), is subject to proteolysis by metalloproteases like ADAMDEC1. Other LRP-interacting proteins are lipoprotein lipase (LPL) and t-PA. For an association study, 165 thrombotic patients with high FVIII levels (from the MAISTHRO, i.e. Main-Isar-thrombosis register) were included. All patients with known causes for high FVIII levels had been previously excluded. The patients were compared with 214 healthy blood donors. Polymorphisms with usually a minor allele frequency >5%, i.e. 24 SNPs and two insertion/deletion polymorphisms of LPL gene, eight SNPs of the t-PA gene, and five SNPs of the ADAMDEC1 gene, were analyzed. Haplotype differences were calculated using PHASE. A new polymorphism in intron 7 of the t-PA gene with a minor allele frequency of 2.2% was identified. Analysis of each SNP by the Cochrane-Armitage trend test did not show any significant association between genotype and disease status. Interestingly, the ADAMDEC1 haplotype (rs12674766, rs10087305, rs2291577, rs2291578, rs3765124) differed between cases and controls (p = 0.04). In particular, the TGTGG haplotype showed a difference. In conclusion, the ADAMDEC 1 haplotype may indicate an underlying mechanism for high FVIII levels. The only moderate linkage disequilibrium may be due to a possible causal polymorphism in distant introns or the promoter region against a polygenic background.

Etiology and VTE risk factor distribution in patients with inferior vena cava thrombosis.

INTRODUCTION: Inferior vena cava (IVC) thrombosis is a rare event and data detailing the underlying etiology are scarce. MATERIALS AND METHODS: Therefore, we reviewed all available cases of IVC thrombosis consecutively registered in the MAISTHRO (MAin-ISar-THROmbosis) database and described the prevalence of VTE risk factors and other conditions contributing to IVC thrombosis development. RESULTS: 53 patients (35 F, 18 M) with IVC thrombosis aged 12 to 79 years were identified. 40 patients (75.5%) developed thrombosis under the age of 45. Local problems, such as IVC anomalies or external venous compression, contributed to the development of thrombosis in 12 cases (22.6%). Lupus anticoagulants (10.9 vs. 2.3%, p=0.013) and malignoma (17.0 vs. 6.4%, p=0.023) were more prevalent in IVC thrombosis patients compared to 265 age and sex matched controls with isolated lower extremity DVT. No difference was identified with regard to inherited thrombophilia or other known VTE risk factors. Symptomatic pulmonary embolism (PE) occurred in 32.1% of IVC thrombosis patients compared to 15.2% of controls (p=0.005). CONCLUSIONS: Local problems such as IVC anomalies and external venous compression, malignancy and the presence of lupus anticoagulants contribute to the risk of IVC thrombosis. The risk of symptomatic pulmonary embolism in the acute setting is high.

New mutations in C1 esterase inhibitor (SERPING1) in a German family with hereditary angioedema.

Hereditary angioedema (HAE) is a genetically dominant clinical disorder characterized by recurrent, acute oedema of the skin or mucosa, usually involving the extremities, face, larynx and gastrointestinal tract. C1 inhibitor (C1inh) deficiency is linked to the development of HAE, either by decrease of its plasma level or presence of a dysfunctional protein. The purpose of this study was to identify the genetic abnormality of C1inh in three patients with HAE (mother and her two children). Analysis was carried out using PCR, and direct sequencing of genomic DNA obtained from whole peripheral blood. DNA sequencing of the eight exons of the C1 esterase inhibitor gene (SERPING1) revealed one mutation and one polymorphism in the mother and the two children. The polymorphism was a heterozygous GTG 458 ATG (Val 458 Met) in exon 8 and the mutation was a one-nucleotide deletion in codon 456 in Exon 8. This frameshift mutation (CTC456(power)TC) leads to a 45 amino acid larger protein with altered protein sequence and should be regarded as the causative defect in the patients. It is concluded, that heterozygous frameshift mutation at exon 8 alters the protein sequence of the C1 esterase inhibitor, leading to inactive protein in all three patients analyzed.

Molecular defects in the ABCA1 pathway affect platelet function.

Platelet function is sensitive to alterations in cholesterol metabolism, and hypercholesterolemia is associated with enhanced platelet reagibility. Atherogenic low-density lipoproteins (LDL), in particular oxidized LDL, activate src-kinase-family-dependent signalling. In contrast, antiatherogenic high-density lipoproteins(HDL) inhibit platelet aggregation and target the phosphatidylinositol phospholipase C (PI-PLC) pathway. Sphingosine 1-phosphate is a major HDL component and may be crucial for downstream reactions of collagen-induced glycoprotein VI signalling and phosphoinositide 3-kinase. The ATP-binding cassette transporter A1 (ABCA1) regulates cell membrane phospholipid and cholesterol homeostasis and their release to lipid-poor apolipoprotein AI to generate prebeta-HDL precursor particles. ABCA1 also interacts with modulators of vesicular trafficking and number and impaired release of dense bodies from platelets. The ABCA1-NH2-terminus-associated Syntaxin-13, a SNARE complex protein, interacts with syntaxin 13-interacting protein (pallidin) whose deficiency leads to impaired platelet granule release from the dense granule Adapter Protein-3 (AP-3)-related pathway. Interestingly, the cholesterol transporter ABCG1 in addition to ABCA1 is another constituent of the AP-3 pathway, and disorders of lysosome-related organelles such as the Hermansky-Pudlack syndrome complex, Chediak-Higashi syndrome and the ceroid lipofuscinoses provide new opportunities to understand AP-3 pathway-related disorders and the irrelation to membrane phospholipid processing. ABCA1 mutations are involved in dysregulated vesicular trafficking from the trans golgi compartment to the plasma membrane, and ABCA1 R1925Q was shown to contribute to Scott syndrome, a phospholipid-processing disorder of missing surface exposure of phosphatidlyserine. The P2Y12 receptor triggers dense granule secretion by downstream effectors including the G-protein-coupled inward rectifier K+ channel-4 (GIRK-4), and we found the sister geneGIRK-3 associated with the ABCA1 protein in macrophages. It is concluded that the presence of ABCA1 and ABCG1 in the AP-3 pathway will have major impact for membrane phospholipid processing and HDL metabolism and their relation to disorders of lysosome-related organelles.

High factor VIII levels in venous thromboembolism show linkage to imprinted loci on chromosomes 5 and 11.

High factor VIII (FVIII) levels are known to be a risk factor for deep venous thrombosis, but the mechanisms responsible for high FVIII levels remain unclear. Here, a new phenotype "FVIII level residuum" (FVIII-R) was defined in order to eliminate the impact of common determinants on FVIII levels. We studied 13 families of patients with thrombosis and reproducibly high FVIII levels of unknown origin. Since familial clustering was evident, we looked for a possible genetic basis. A genome scan was performed with 402 evenly spaced microsatellite markers. A quantitative linkage analysis using variance component methods showed suggestive evidence for linkage of FVIII-R with a locus on chromosome 8 (logarithm of odds [LOD] = 2.1). In addition, we performed parametric exploratory linkage analysis of dichotomized FVIII-R, taking a parent-of-origin effect into account. Single-trait-locus MOD-score analysis showed suggestive evidence for linkage under an imprinting model at chromosomes 5 and 11. Furthermore, a 2-trait-locus analysis under a multiplicative model for the loci of chromosomes 5 and 11 yielded a remarkable LOD of 4.44. It confirmed the finding of paternal imprinting, obtained by single-trait-locus analysis, at both loci. Our results suggest that high FVIII levels in venous thromboembolism represent a complex trait caused by several genetic factors.

High factor VIII (FVIII) levels in venous thromboembolism: role of unbound FVIII.

Theoretically, von Willebrand factor (VWF) should be capable of binding all factor VIII (FVIII), but an unbound FVIII (uFVIII) plasma fraction remains. In patients' status post deep-vein thrombosis (DVT), an altered uFVIII fraction and high FVIII levels might be indicative of dysfunctional FVIII regulation. Out of 928 consecutive DVT patients, 321 were found to have high FVIII levels. After excluding 183 patients with known causes for high FVIII levels, plasma samples with unexplainably high FVIII levels were available from 84 patients. To capture the FVIII-VWF-complex, superparamagnetic polystyrene beads with covalently attached streptavidin were coated with biotinylated anti-rabbit Ig and incubated with rabbit anti-human VWF-Ig. Slowly thawed plasma samples were added to cooled beads, which were then separated by a magnetic particle concentrator. The uFVIII fraction was calculated by dividing the FVIII activity in the supernatant of the FVIII-VWF-complex-free sample by the FVIII activity in the supernatant of the control sample. Additionally, the VWF residuum in the supernatant was determined. Compared to age- and sex-matched blood donors, thrombosis patients showed a significantly higher plasma FVIII/VWF ratio (median: 1.3 vs. 1.0, p<0.001). uFVIII fraction data were adjusted for VWF residuum. After forward stepwise logistic regression, uFVIII had an odds ratio of 0.48 (95% CI 0.34-0.65), i.e. the uFVIII fraction was reduced in thrombosis patients. Analysis of covariance confirmed these results: In thrombosis patients, the estimated mean of the uFVIII fraction was significantly lower (6.34% vs. 7.58%, p<0.001). In conclusion, thrombosis patients with high FVIII levels showed a higher FVIII/VWF ratio, similar to mice with defective FVIII clearance. The clearly reduced uFVIII fraction lends further support to the hypothesis of a modified FVIII clearance.

CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 genotypes in patients with deep-vein thrombosis.

The role of methylenetetrahydrofolate reductase (MTHFR) TT677 genotype, cystathionine beta-synthase (CBS) 844ins68 mutation and endothelial cell protein C receptor (EPCR) 4031ins23 in the development of deep-vein thrombosis (DVT) was investigated in 300 consecutive DVT patients and 410 healthy blood donors. MTHFR TT677 was found in 40 (13.3%) patients and in 59 (14.4%) controls (OR 0.92; 95% C.I. 0.54-1.41); CBS 844ins68 in 20 (6.7%) patients and in 56 (13.7%) control subjects (OR 0.45; 95% C.I. 0.27-0.77); and the combination of MTHFR TT677 with CBS 844ins68 in 4 (1.3%) patients and in 7 (1.7%) controls (OR 0.78; 95% C.I. 0.23-2.68). Logistic regression analysis did not show a further increase of risk for MTHFR TT677 or CBS 844ins68 in combination with the factor V Leiden or the prothrombin gene G20210A mutations. The EPCR 4031ins23 was observed in 2 patients (0.66%) and none of the controls. In conclusion, MTHFR TT677 does not appear to be an important risk factor for DVT, EPCR 403ins23 seems to be very rare, its role in the development of DVT unclear. A putative protective effect of CBS 844ins68 should be further investigated.