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Rationale: A reduced rate of myocardial infarction has been reported in patients with atrial fibrillation treated with FXa (factor Xa) inhibitors including rivaroxaban compared with vitamin K antagonists. At the same time, low-dose rivaroxaban has been shown to reduce mortality and atherothrombotic events in patients with coronary artery disease. Yet, the mechanisms underlying this reduction remain unknown., Objective: In this study, we hypothesized that rivaroxaban's antithrombotic potential is linked to a hitherto unknown rivaroxaban effect that impacts on platelet reactivity and arterial thrombosis., Methods and Results: In this study, we identified FXa as potent, direct agonist of the PAR-1 (protease-activated receptor 1), leading to platelet activation and thrombus formation, which can be inhibited by rivaroxaban. We found that rivaroxaban reduced arterial thrombus stability in a mouse model of arterial thrombosis using intravital microscopy. For in vitro studies, atrial fibrillation patients on permanent rivaroxaban treatment for stroke prevention, respective controls, and patients with new-onset atrial fibrillation before and after first intake of rivaroxaban (time series analysis) were recruited. Platelet aggregation responses, as well as thrombus formation under arterial flow conditions on collagen and atherosclerotic plaque material, were attenuated by rivaroxaban. We show that rivaroxaban's antiplatelet effect is plasma dependent but independent of thrombin and rivaroxaban's anticoagulatory capacity., Conclusions: Here, we identified FXa as potent platelet agonist that acts through PAR-1. Therefore, rivaroxaban exerts an antiplatelet effect that together with its well-known potent anticoagulatory capacity might lead to reduced frequency of atherothrombotic events and improved outcome in patients.

Background: Aortic root ectasia might induce hemostatic disorders in patients with Marfan syndrome (MFS) via altered blood flow and rheology. The aim of this study was to explore the hemostasis in patients with MFS compared with healthy controls., Methods: In this cross-sectional case-control study we included patients with verified MFS (n=51) and sex- and age-matched healthy controls (n=50). Main criteria were the aortic root in echocardiography and cardiac magnetic resonance imaging (MRI), and the coagulation status., Results: When compared with healthy controls, patients with MFS showed significantly increased diameters of the aortic roots (43.0±7.72 vs. 28.8±3.74 mm, P<0.001) and aortic Z-scores (4.36±2.77 vs. 0.948±1.09, P<0.001), considerably higher values of Multiplate ® tests (e.g., MP-ADP: 878.4±201.7 vs. 660.4±243.6 AU*min, P<0.001) and PFA-100 ® tests (PFA Col/ADP: 102.5±45.5 vs. 91.1±46.2 s, P<0.05), PTT (30.0±3.91 vs. 28.7±2.50 s, P<0.05) and D-dimers (0.488±0.665 vs. 0.254±0.099 mg/L, P<0.001). In MFS von Willebrand factor (VWF) activity (81.9%±41.8% vs. 106.3%±41.5%, P<0.05) and antigen (93.8%±43.9% vs. 118.8%±47.8%, P<0.05) and factor VIII activity (108.9%±29.6% vs. 126.7%±28.4%, P<0.05) were reduced. Significant positive correlations were found between aortic diameters and D-dimers (all P<0.05), as well as PFA Col/ADP (all P<0.01) in MFS patients. Factor VIII activity correlated significantly negatively with the diameter of the aortic root in MFS (r=-0.55, P<0.05)., Conclusions: In conclusion, our study reveals hemostatic deviations in patients with MFS. Further studies are necessary to understand the causal relationship and the exact pathomechanism., Competing Interests: Conflicts of interest: The authors have no conflicts of interest to declare., (2019 Cardiovascular Diagnosis and Therapy. All rights reserved.)

Adults with cyanotic congenital heart diseases (CCHD) have a higher risk for bleeding, but also for thrombosis. Rotational thromboelastometry (RT), using tissue factor (EXTEM), a contact activator (INTEM) or cytochalasin (FIBTEM), assesses coagulation by determining the time to initiation of clotting (CT) and clot firmness (MCF) including platelet-fibrin-interaction. The aim of this study was to evaluate RT and whole blood impedance aggregometry (IA) in CCHD compared with a control group without chronic cyanosis (NCCHD). These were used to establish normal reference ranges. We prospectively included 124 patients (76 CCHD, 48 NCCHD). Mean oxygen saturation in CCHD was 81.5%, and 98% in NCCHD (p <0.001). Fifty-five CCHD and 1 NCCHD had pulmonary hypertension. Eisenmenger syndrome was present in 39 CCHD (51.3%). Hemoglobin, hematocrit, and reticulocyte levels were significantly higher in CCHD, and they also showed more thrombocytopenia. Platelet aggregation was under normal range in 89.5% of CCHD after triggering with ADP, in 85.5% after triggering with arachidonic acid (ASPI) and in 73.7% after TRAP-6. RT showed significantly longer clotting times and reduced clot firmness in both EXTEM and INTEM tests. FIBTEM-MCF was also significantly reduced. Moderate inverse correlation was found between platelet count and erythrocytes (r = -0.608, p <0.001). Significant correlations were found between platelet number and RT-parameters as well as with all IA parameters. In conclusion, according to RT and IA, CCHD present hypocoagulable disorders. No signs of hypercoagulability were found., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Five contigs of the 5' part of the immunoglobulin kappa locus (F. Röschenthaler et al., Eur. J. Immunol. 1999. 29: 2065 - 2071) have been linked by cosmid clones prepared from bacterial artificial chromosomes (BACs) and by PCR. One of the previously defined contigs which contains three pseudogenes (Z7) was shown by fluorescence in situ hybridization to be located near the kappa locus on chromosome 6, but not within the locus; the three Vkappa genes are therefore now classified as orphons. A Vkappa9 / 10 gene, which was sequenced previously, was now localized within the locus, and two additional Vkappa genes were identified, a potentially functional Vkappa24 gene and a pseudogene of the Vkappa9 / 10 family. This brings the number of localized and sequenced Vkappa genes in the locus to 140; 75 of them are functional, 21 potentially functional and 44 pseudogenes. The 5' part of the kappa locus is now one contig of 1.88 megabase (Mb); it comprises 82 Vkappa genes. Other contigs of the locus are 65 kb, 105 kb and 1.04 Mb in size and contain 2, 5 and 51 Vkappa genes, respectively. The contigs are separated by three gaps of 10 - 40 kb each. Detailed restriction maps and other structural details of the kappa locus are deposted in the Internet at http://www.med. uni-muenchen.de / biochemie / zachau / kappa.htm.

The 5' region of the mouse kappa locus comprises 63 Vkappa genes in six contigs of together 1.5 Mb, including one which links the region to the central part of the locus. The structures of the contigs were established by detailed restriction mapping of cosmid clones prepared from libraries of mouse C57BL/6 DNA and of yeast and bacterial artificial chromosomes (YACs, BACs with mouse DNA inserts). Pulsed-field gel electrophoresis of yeast artificial chromosome digests indicated that the gaps between the contigs were 10 to 60 kb, comprising together about 160 kb. The region of the kappa locus described here contains Vkappa1, Vkappa2, Vkappa9/10, Vkappa11, Vkappa12/13, Vkappa20, Vkappa24, Vkappa32, Vkappa33/34 and Vkappa38C genes as well as the VkappaRF gene and, towards the center of the locus, a number of Vkappa4/5 genes. Near the 5' end of the locus interspersed alpha-tubulin gene-like sequences were found. At its 3' side the region borders on the Vkappa4/5 contigs of the central region of the locus which is described in the accompanying report (Eur. J. Immunol. 1999. 29: 2057-2064). Structural details are to be found in the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section the main features of the structure of the mouse kappa locus are summarized.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.

At the present state of analysis the central part of the kappa locus comprises four contigs of together 1.2 Mb and contains 55 Vkappa genes. It is flanked by the 3' part of the locus with 22 Vkappa genes in 0.4 Mb (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) and the 5' part with 63 Vkappa genes in six contigs of together 1.5 Mb (F. Röschenthaler et al., accompanying report). The 5' and the central regions have one large contig in common. A part of the central region is linked to the 3' region resulting in a 1.1-Mb contig. The structure of the contigs was established mainly by the analysis of overlapping cosmid clones derived from genomic DNA and yeast and bacterial artificial chromosomes (YACs and BACs) and by PCR techniques. Pulsed-field gel electrophoresis of YAC digests indicated that three gaps between the contigs of the central region are 10-40 kb in size, comprising together about 90 kb. Internal duplications in this part of the locus and rearranged YACs were the major problems of the structural work. Structural details are to be found on the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section of the report the mouse kappa locus is compared to the human one and some aspects of the evolution of the kappa locus are discussed.

A detailed restriction map of a 430-kb contig comprising the single Ckappa, the 5 Jkappa and the adjoining 22 Vkappa gene segments is presented. The first 12 Vkappa genes following the JkappaCkappa region belong to the Vkappa21 family, the subsequent ones to the closely related families Vkappa8 and Vkappal 9/ 28. Previous difficulties in cloning all Vkappa21 genes can now be explained by the presence of a duplicated region in this part of the locus. The structure was established by analysis of yeast artificial chromosome, bacterial artificial chromosome and cosmid clones and by the so-called long template PCR technique. The distance between Ckappa and the proximal Vkappa21 gene is 22 kb and the average distances between the Vkappa genes are about 20 kb. Of the 12 Vkappa21 genes 5 were sequenced for the first time and 8 of the 12 genes were found to be expressed. Of the 10 Vkappa8 and Vkappa19/28 germline genes 9 are new; expression products of 8 of the 10 genes were known. The known 5', 3' polarities allow to specify for the 22 Vkappa genes whether they are rearranged to the JkappaCkappa element by a deletion or an inversion mechanism. Also the formation of interesting rearrangement products in classical cell lines as MPC11, MOPC41 and PC 7043 can be explained now. The non-Vkappa sequence L10 whose rearrangement by inversion has been described earlier (Hoechtl and Zachau, Nature 1983. 302: 260-263) was now localized downstream of JkappaCkappa.

Although numerous solitary germ-line V kappa genes and two small V kappa contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the kappa locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different V kappa gene families. Ninety-one V kappa gene-containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the kappa locus is known to reside; no evidence for the existence of dispersed V kappa genes (orphons) was obtained. Eighty-five strong hybridization signals were assigned to distinct V kappa gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one V kappa gene family ("clusters") and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional V kappa genes which are known to exist will have to be isolated from other gene libraries of the same mouse Ig kappa haplotype.

Two problems in defining the germline repertoire of immunoglobulin kappa genes were investigated. One concerns putative transposed V kappa genes (orphons), the other one weak hybridization signals which may or may not turn out to be V kappa genes (UHOs). It was shown by sequencing that the three V kappa genes Z2, Z3 and Z4 are very closely related to the Z1 and V118 genes and to two other genes which had been localized on chromosomes 1 and 22, i.e. outside the kappa locus on chromosome 2. It is therefore likely that also the Z2-Z4 genes are orphons and not part of the kappa locus. Two UHOs turned out not to contain V kappa-like structures. This together with previous results makes it likely that we have detected all germline V kappa genes with the available hybridization probes.