Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.     

Delayed activation of quiescent donor hematopoietic stem cells in the host marrow cavity by anti-host monoclonal antibody

To understand the mechanisms controlling hematopoietic engraftment in untreated, normal recipients, we investigated the fate of parental, donor hematopoietic stem cells after apparent graft failures in unconditioned F1 hybrid recipient mice. By administering an anti-host H- 2K monoclonal antibody, which targets host cells but spares the donor, we found that chimerism could be induced by delayed conditioning in animals with apparent graft failure. Engraftment kinetics in the host were followed by typing individual colony forming unit-- granulocyte/macrophage (CFU-GM) colonies for their origin and showed that parental cells, which were otherwise virtually absent, become promptly detectable within the marrow cavity after antibody administration. Marrow transfers to secondary hosts suggested that parental stem cells were present in the marrow of the untreated recipients. These findings establish that the elimination of all parental cells cannot account for the absence of peripheral blood chimerism in the unconditioned F1 hybrid recipient. Thus, viable and functional donor stem cells, which remain quiescent in the host marrow, can be activated by a selective conditioning regimen and can rescue an apparent graft failure. The selective activation in vivo of marked stem cells in an unirradiated microenvironment may be a useful system to study the regulation of cellular proliferation within the marrow cavity.     

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P- selectin-mediated binding of platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P- selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.     

Studies on the transmission of Venezuelan equine encephalitis virus by Colombian simuliidae (Diptera)

The ability of Simulium mexicanum and Simulium metallicum to serve as biological or mechanical vectors of an enzootic and an epizootic strain of Venezuelan equine encephalitis (VEE) virus was examined. Guinea pigs were inoculated with the epizootic Cordoba strain or the enzootic RPVP407 strain of VEE virus. Wild-caught adult Simuliidae were fed on the viremic guinea pigs and the virus content of groups of flies was determined at daily intervals post-engorgement to test for viral replication. Flies were refed on suckling mice at greater than or equal to 8 days post-engorgement to test for biological transmission. Other flies were interrupted while feeding on viremic guinea pigs and refed on suckling mice to test for mechanical transmission. Neither S. mexicanum nor S. metallicum appear to be efficient vectors of either strain of VEE virus, although occasional mechanical transmission was obtained. Titers of virus in flies decreased rapidly after engorgement and from 3-12 days post-engorgement virus was detected only in 5%-25% of both species of flies. Although earlier field evidence implicated both S. mexicanum and S. metallicum as vectors of epizootic VEE, we conclude that it is highly unlikely that they play an important role as vectors of the virus in nature.     

Aminoglycoside-associated hypomagnesaemia in children with cystic fibrosis

Hypomagnesaemia in children with cystic fibrosis (CF) is under-recognized. We report a child with CF who developed significant hypomagnesaemia following intravenous (i.v.) treatment with aminoglycosides for exacerbations of Pseudomonas aeruginosa infection. Three additional cases have also been observed. Investigations in two patients have revealed excessive renal loss of magnesium. It is postulated that renal tubular damage secondary to the cumulative effects of repeated courses of aminoglycosides resulted in hypomagnesaemia, and we suggest screening for this problem by monitoring serum magnesium regularly in all patients with CF receiving multiple courses of aminoglycosides.     

Differentiation and proliferation of spermatogonial stem cells using a three-dimensional decellularized testicular scaffold: a new method to study the testicular microenvironment in vitro

International Urology and Nephrology - Successful in vitro transplantation of spermatogonial stem cells (SSCs) demands effective culture systems for SSCs proliferation and differentiation. Natural...     

Liver transplant recipients: portable duplex US with correlative angiography

Twenty patients, aged 4 months to 58 years, were evaluated for liver transplantation by duplex sonography, and 15 transplantations were completed; 42 postoperative examinations were performed. Sonographic findings were correlated with seven preoperative and five postoperative angiographic evaluations. Preoperative duplex US findings included tumors, portal vein occlusion, varices, biliary obstruction, and variant vascular anatomy. Postoperative findings included hepatic artery occlusion, portal vein occlusions (one with cavernous transformation), portal vein stenosis, biliary obstruction, intrahepatic and extrahepatic fluid collections, and air in the portal vein due to ischemic bowel. Use of angiography allowed confirmation of the vascular abnormalities and demonstrated evidence of rejection in patients with normal Doppler waveforms. Duplex sonography is a valuable portable technique for evaluating these patients and can be used in triage of patients requiring angiography.