Discrepant platelet and plasma von Willebrand factor in von Willebrand disease patients with p.Pro2808Leufs*24.

Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY: Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

Non-genomic activities of retinoic acid receptor alpha control actin cytoskeletal events in human platelets.

UNLABELLED: Essentials Platelets employ proteins/signaling pathways traditionally thought reserved for nuclear niche. We determined retinoic-acid-receptor alpha (RARα) expression and function in human platelets. RARα/actin-related protein-2/3 complex (Arp2/3) interact via non-genomic signaling in platelets. RARα regulates Arp2/3-mediated actin cytoskeletal dynamics and platelet spreading. SUMMARY: Background Platelets utilize proteins and pathways classically reserved for the nuclear niche. Methods We determined whether human platelets express retinoic-acid-receptor family members, traditionally thought of as nuclear transcription factors, and deciphered the function of RARα. Results We found that RARα is robustly expressed in human platelets and megakaryocytes and interacts directly with actin-related protein-2/3 complex (Arp2/3) subunit 5 (Arp2/3s5). Arp2/3s5 co-localized with RARα in situ and regulated platelet cytoskeletal processes. The RARα ligand all-trans retinoic acid (atRA) disrupted RARα-Arp2/3 interactions. When isolated human platelets were treated with atRA, rapid cytoskeletal events (e.g. platelet spreading) were inhibited. In addition, when platelets were cultured for 18 h in the presence of atRA, actin-dependent morphological changes (e.g. extended cell body formation) were similarly inhibited. Using in vitro actin branching assays, RARα and Arp2/3-regulated complex actin branch formation was demonstrated. Consistent with inhibition of cytoskeletal processes in platelets, atRA, when added to this branching assay, resulted in dysregulated actin branching. Conclusion Our findings identify a previously unknown mechanism by which RARα regulates Arp2/3-mediated actin cytoskeletal dynamics through a non-genomic signaling pathway. These findings have broad implications in both nucleated and anucleate cells, where actin cytoskeletal events regulate cell morphology, movement and division.

Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA.

Diannexin, an annexin A5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.

BACKGROUND: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life. OBJECTIVES: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation. METHODS: The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser-induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. RESULTS: Diannexin and annexin A5 bind PS with K(D) values of 0.6 and 5 nm, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nm (P<0.05-0.001 compared with control), and reduced platelet accumulation at 1 µg g(-1) (P<0.05) and activation at 0.25 µg g(-1) (P<0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 µg g(-1) (P<0.01). CONCLUSIONS: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.

Superoxide dismutase halts cycling of murine erythroid progenitor cells prior to S phase in vitro and possibly in vivo.

Mice of the C57BL/6 (B6) strain show a much lower proportion of marrow erythroid progenitor cells (BFU-E) in DNA synthesis in vivo than mice of the congeneic B6S strain. However, when assayed in vitro marrow cells from both strains show high proportions of BFU-E in S-phase. BFU-E from normal B6 mice have been previously shown to be specifically inhibited from entering S-phase in vitro by the antioxidant enzyme superoxide dismutase (SOD), however, in this study we have found that BFU-E taken from the marrow of B6S mice or B6 mice which have been subjected to bleeding are insensitive to SOD inhibition in vitro. Comparisons of results from in vivo and in vitro cycling assays done with cells from both strains indicate that a large proportion of marrow BFU-E in normal B6 mice are halted in the pre-S portion of the cell cycle in vivo, and these halted cells are prevented from going into S-phase in vitro by SOD. The insensitivity to SOD inhibition shown by BFU-E from B6S and bled B6 mice can be attributed to the absence of accumulation of SOD-susceptible cells in pre-S phase in these mice in vivo, and there is evidence to suggest that the difference in BFU-E cycling seen in vivo may be due to interactions between SOD and factors which stimulate cycling of BFU-E.

Superoxide dismutase as an inhibitor of erythroid progenitor cell cycling.

C57Bl/6 (B6) mice and mice of a congeneic strain, B6S, differ in the proportions of erythroid progenitor cells (BFU-E) typically seen in DNA synthesis in in vivo cell suicide assays, and bone marrow supernatants (MS) prepared from B6 mice can inhibit BFU-E cycling in vitro. Using in vitro BFU-E DNA synthesis assays and a model system of BFU-E in culture (DA-1 cells) as screening methods for the detection of inhibitors of BFU-E cycling, we have purified the protein that is apparently responsible for the inhibitory effects of MS on progenitor cells and that is also an antagonist of the stimulatory effects of interleukin-3 (IL-3) on DA-1 cell proliferation in culture. We have identified this protein as the Cu,Zn-containing form of the antioxidant enzyme superoxide dismutase (SOD), which is normally present in large amounts in erythrocytes. MS from B6S mice does not inhibit BFU-E DNA synthesis. However, measurements of SOD activity showed no differences between B6 and B6S mice; thus the difference between the effects of B6S-MS and B6-MS is not due to differences in the levels of SOD present. The inhibitory effects of SOD on BFU-E in vitro are opposed by the stimulatory effects of IL-3 in a dose-dependent manner, and similar interactions between stimulatory and inhibitory factors also appear to determine the effects of mouse-derived preparations on erythroid cells. If the interactions seen in vitro are applicable to the state in vivo, SOD may be a constitutive inhibitor of erythroid progenitor cell cycling in mice, acting in opposition to stimulatory factors whose expression varies in response to genetic and physiological influences.

Superoxide dismutase specifically inhibits erythroid cell DNA synthesis and proliferation.

The antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for erythroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.

Purification of an inhibitor of erythroid progenitor cell cycling and antagonist to interleukin 3 from mouse marrow cell supernatants and its identification as cytosolic superoxide dismutase.

We have isolated a protein from media conditioned by a murine marrow-derived cell line (PB6) and from mouse marrow supernatants that antagonizes interleukin 3-dependent proliferation of cells in culture and reversibly inhibits DNA synthesis of erythroid progenitor cells (BFU-E) in vitro. This protein, p16 (monomer Mr = 16 kD on SDS-PAGE), was purified to homogeneity and amino acid sequencing of a polypeptide fragment yielded a sequence identical to that of murine cytosolic Cu,Zn-containing superoxide dismutase (SOD). The identification of p16 as SOD was confirmed by the detection of SOD enzymatic activity in pure p16 fractions, and when a commercial human erythrocytic SOD preparation was tested it showed the same cell inhibitory activities as p16. These observations show that superoxide dismutase is able to affect the cycling and growth factor responses of hematopoietic cells, activities that have not previously been associated with this enzyme.

The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay.

The suggestion that compensation for overabundant mRNA of the genes for Saccharomyces cerevisiae ribosomal protein (r-protein) L3, L29, or rp59 occurs by translation repression has been reinvestigated. First, analysis of the distribution of these three mRNAs in polysome profiles revealed no differences between normal and mRNA-overproducing strains, indicating that initiation of r-protein translation is not repressed under conditions of mRNA overaccumulation. Second, experiments involving radioactive pulse-labeling of proteins were done by using a modified method of data collection and analysis that allows quantitation and correction for fast decay during the pulse. These measurements revealed that the synthesis rate of the three r-proteins is increased when their mRNA levels are elevated and that their decay rate is also high, with half-lives ranging from a fraction of a minute to more than 10 min. We conclude that accumulation of excess r-protein mRNA has no effect on translation rate; rapid decay of protein during the course of the labeling period can account for the apparent discrepancy between mRNA levels and protein synthesis rates. Yeast r-proteins, when produced in excess, are among the most rapidly degraded proteins so far described.