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.     

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.     

Visual discrimination and short-term memory for random patterns in patients with a focal cortical lesion

Visual discrimination and short-term recognition memory for computer- generated random patterns were explored in 23 patients with a postsurgical lesion in one of the cortical hemispheres. Their results are compared with those of 23 age-matched volunteers. In a same- different forced-choice discrimination task, d' and log beta (measures of sensitivity and bias), as well as reaction time (RT) were determined. All participants viewed patterns defined either by luminance contrast or isoluminant red-green color contrast, the amplitude of which was adjusted to be 10 times the respective detection threshold level. Block patterns consisting of a 6 x 6 matrix of light and dark (red and green) checks were randomly configured on each presentation. They were presented in pairs, randomly in two visual quadrants for a duration of 200 msec. Three presentation conditions were used: simultaneous presentation of reference and test stimulus, sequential presentation with a short delay (interstimulus interval, ISI = 3 s), and sequential presentation with a long delay (ISI = 6 s). The results indicate that patients with a lesion in the occipitotemporal cortex, the superior temporal cortex and the frontal cortex were significantly impaired on both luminance-contrast and color-contrast pattern discrimination. Patients with damage in the anterior inferotemporal cortex showed no overall impairment. The results suggest that performance in visual discrimination and recognition memory tasks rely on distributed neural processes with more than one neocortical location.      

Synergism between parathyroid hormone and interleukin 1 in stimulating bone resorption in organ culture

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1 alpha and interleukin 1 beta were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.     

Progression of occlusive disease following femorofemoral crossover bypass graft

Progression of distal disease is considered the most common cause of femorofemoral artery cross-over bypass graft (FFBPG) failure. Twenty-seven patients with patent grafts (mean 53 months) were evaluated with segmental Doppler and duplex scan arterial studies for evidence of disease progression. In the early postoperative period (compared with preoperative levels), 26 patients (95.3%) showed a significant improvement (>0.1) in the recipient limb ankle-brachial index (ABI) (mean increase of 0.38; SD=0.24) and/or ankle spectral arterial waveform. However, there was a statistically significant decrease (p=0.0001) in the donor limb ABI, and 12 patients (44.4%) had a >0.1 deterioration. On long-term follow-up (compared with preoperative levels) this difference was no longer significant (p=0.49); only seven donor limbs remained with a >0.1 decrease in ABI. The recipient limbs maintained a significant improvement (>0.1) in the ABI compared to preoperative levels (p<0.0001; mean of 0.39; SD=0.16) except for three limbs that had decreased by 0.1. However, eight patients (29.6%) developed an increase in their donor common femoral artery acceleration time >133 msec and/or increased blood flow velocity without a simultaneous significant decrease in their recipient limb ABI. In the latter group the preoperative donor limb common femoral artery acceleration time and ABI and the immediate postoperative change in donor limb ABI were not significantly different (p>0.05) than in the remaining patients. These data suggest that there is a significant but transient decrease in donor limb ABI following FFBPG, that long-term changes in ABIs are symmetric in both limbs, and that inflow surveillance with duplex scanning and acceleration time measurement may allow early detection of hemodynamically significant inflow lesions prior to graft failure. Presented at the Seventeenth Annual Meeting of the Peripheral Vascular Surgery Society, Chicago, Ill., June 7, 1992.     

HIV/AIDS infection control enforcement: a comparison between Nigeria and the United States

We examined differences in approaches to HIV-related infection control practices in two university teaching hospitals in the United States and Nigeria. Health care workers (n = 202 in Nigeria and 186 in the USA) responded to a previously validated measure of infection control practices. There were significant differences in the estimated probability of treating a person with HIV disease (higher in USA), and a greater probability of peer ridicule as a way of enforcing group norms on infection control in Nigeria. Peer enforcement of norms was significantly lower in the USA. In both countries, more precautions would be taken if it was known that the patient was HIV infected. Infection control practices were more likely to be followed in the USA compared with Nigeria if they were praised for this activity, if appropriate facilities (sharps containers, gloves, etc.) were nearby, and if they felt that infection control procedures were effective. These data point to the importance of normative social pressures in Nigeria and of knowing the patient is HIV infected in the USA and feeling that infection control procedures are effective ways of avoiding occupational HIV infection. The role of normative pressures and assumptions about HIV infection status as well as cues and availability of facilities for infection control appear to differ between these health care workers in Nigeria and the USA.