Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.

Thrombin generation and secretion of platelet Factor 4 during blood clotting.

We have studied the platelet release reaction and thrombin generation during the spontaneous clotting of whole blood in vitro. Both thrombin formation and secretion of platelet Factor 4 were detected at least 12 min before clotting (clotting time, 22--26 min). Initially, at low thrombin concentrations (2--5 ng/ml), there is a small increase in plasma platelet Factor 4 (less than 1% of the amount present in serum). This is followed by a gradual increase in both platelet Factor 4 and thrombin concentrations over a 12 to 20-min interval. Finally, 5 min 5 before clotting, there is a rapid increase in both thrombin generation and platelet secretion. Thus, we have shown that the release of platelet Factor 4 is a prolonged reactoin and the extent to which it occurs parallel thrombin generation. It is only when thrombin concentrations are high (45--90) ng/ml)--during the period of clot formation--that the major part of platelet Factor 4 secretion occurs. Release of platelet Factor 4, like fibrin formation, occurs in the last step of in vitro coagulation.

Relationship between secretion of platelet Factor 4 and thrombin generation during in vitro blood clotting.

We have studied the effects of both impaired prothrombin activation and direct inhibition of thrombin on the platelet release reaction in clotting blood to determine the role of thrombin in this process. In blood from two patients with congenital Factor V deficiency, prothrombin activation during spontaneous in vitro clotting was delayed and decreased. Secretion of platelet Factor 4 was also delayed and was detected only after thrombin formation was initiated. Addition of a small amount of normal plasma to the patients' blood in vitro corrected the abnormalities in both thrombin formation and the platelet release reaction in parallel fashion. A delay in the onset of secretion of platelet Factor 4 was also observed when thrombin generated in normal blood during spontaneous in vitro clotting was inhibited by either purified hirudin or anti-thrombin Fab. These observations suggest that thrombin is the essential stimulus for platelet secretion during in vitro blood clotting. The effect of inhibitors of the platelet release reaction on prothrombin activation during in vitro blood clotting was also studied. When either prostacyclin or the combination of prostaglandin E(1) and N(6)O(2')-dibutyryl cyclic AMP was added, secretion of platelet Factor 4 was inhibited 85-95%. We were unable to detect any inhibition of initiation of prothrombin activation or inhibition of that part of thrombin generation associated with clotting. These results indicate either that the platelet release reaction may not be required for the initiation of prothrombin activation or only a very limited amount of secretion may be necessary for normal generation of thrombin to occur.

Immunoglobulin G is a platelet alpha granule-secreted protein.

It has been known for 27 yr that blood platelets contain IgG, yet its subcellular location and significance have never been clearly determined. In these studies, the location of IgG within human platelets was investigated by immunocytochemical techniques and by the response of platelet IgG to agents that cause platelet secretion. Using frozen thin-sections of platelets and an immunogold probe, IgG was located within the alpha-granules. Thrombin stimulation caused parallel secretion of platelet IgG and two known alpha-granule proteins, platelet factor 4 and beta-thromboglobulin, beginning at 0.02 U/ml and reaching 100% at 0.5 U/ml. Thrombin-induced secretion of all three proteins was inhibited by prostaglandin E1 and dibutyryl-cyclic AMP. Calcium ionophore A23187 also caused parallel secretion of all three proteins, whereas ADP caused virtually no secretion of any of the three. From these data and a review of the literature, we hypothesize that plasma IgG is taken up by megakaryocytes and delivered to the alpha-granules, where it is stored for later secretion by mature platelets.

Neuromuscular transmission in rheumatoid arthritis, with and without penicillamine treatment.

Electroneuromyographic studies were performed on patients with rheumatoid arthritis, some of whom were received penicillamine, to determine whether a subclinical defect of neuromuscular transmission existed. There were no significant differences between patients and controls with respect to nerve conduction studies or repetitive ulnar nerve stimulation. Four patients (three receiving penicillamine) demonstrated mild neurogenic changes distally on needle electromyography. Mean jitter was slightly higher for patients receiving penicillamine than in other patients or controls, but the differences were not significant. No significant correlations existed between of the studies and daily, cumulative, or average penicillamine dosage. A significant positive correlation (p less than 0.001) existed between jitter and duration of disease in patients receiving penicillamine. Results were consistent with the hypothesis that penicillamine predisposes certain individuals to develop myasthenia gravis rather than interfering directly with neuromuscular transmission.

Asymptomatic sensorimotor polyneuropathy in workers exposed to elemental mercury.

Neurologic and electrodiagnostic evaluations and urine mercury level determinations were performed on 138 chlor-alkali plant workers, some of whom were chronically exposed to inorganic mercury vapor. Eighteen subjects had a mild polyneuropathy on clinical examination. These subjects had significantly (p less than 0.05) elevated urine mercury indexes, reduced sensation on quantitative testing, prolonged distal latencies with reduced sensory evoked response amplitudes, and increased likelihood of abnormal needle electromyography compared with the remaining 120 subjects. Similar results were found for subgroups matched by sex and age. We conclude that elemental mercury exposure is associated with a sensorimotor polyneuropathy of the axonal type; the degree of neurologic impairment appears related to the magnitude of exposure.

Massive hepatomegaly following splenectomy for myeloid metaplasia. Case report and review of the literature.

Massive compensatory hepatic myeloid metaplasia occurs infrequently after splenectomy for myeloid metaplasia, and it usually develops gradually over several years if it does occur. In this report, a patient is described in whom massive hepatomegaly and evidence of portal hypertension developed within three months of splenectomy. Early evidence for rapid hepatic growth was present at a second laparotomy three weeks after the first surgery, and probably should have triggered initiation of chemotherapy. Indications for splenectomy in these patients, surgical morbidity, and response to splenectomy are discussed.

Visualization of significant ERD/ERS patterns in multichannel EEG and ECoG data.

OBJECTIVES: Analysis of event-related desynchronization (ERD) and event-related synchronization (ERS) often requires the investigation of diverse frequency bands. Such analysis can be difficult, especially when using multichannel data. Therefore, an effective method for the visualization of event-related changes in oscillatory brain activity is required. METHODS: A bootstrap-based method is presented which gives time-frequency maps showing only significant changes of ERD or ERS in predetermined frequency bands. RESULTS: Examples from an electroencephalographic study and an electrocorticographic study are shown. The results demonstrate how easily reactive channels and their spatio-temporal and frequency-specific characteristics can be identified by means of this method. CONCLUSIONS: The proposed method is a simple but effective way to visualize significant ERD/ERS patterns.

Spatiotemporal patterns of beta desynchronization and gamma synchronization in corticographic data during self-paced movement.

OBJECTIVE: To study the spatiotemporal pattern of event-related desynchronization (ERD) and event-related synchronization (ERS) in electrocorticographic (ECoG) data with closely spaced electrodes. METHODS: Four patients with epilepsy performed self-paced hand movements. The ERD/ERS was quantified and displayed in the form of time-frequency maps. RESULTS: In all subjects, a significant beta ERD with embedded gamma ERS was found. CONCLUSIONS: Self-paced movement is accompanied not only by a relatively widespread mu and beta ERD, but also by a more focused gamma ERS in the 60-90 Hz frequency band.

Platelet factor 4 does not promote von Willebrand factor binding to human platelets.

Ristocetin, protamine and Polybrene promote factor VIII:vWF binding and agglutination of formalinized platelets. It has been suggested that these polycations neutralize platelet negative surface charges and promote the attachment of VIII:vWF to platelets. Platelet factor 4 (PF4), protamine, and Polybrene inhibit heparin activity by neutralizing heparin negative charges. We tested the hypothesis that PF4, which is bound to the platelet surface after platelet activation and secretion, could promote the binding of VIII:vWF and subsequent platelet agglutination. Purified PF4 in concentrations comparable to those of ristocetin did not agglutinate formalinized platelets or induce the disappearance of VIII:vWF from the suspending plasma. Platelets were thrombin-treated in order to induce the release of PF4, and then formalinized and resuspended in normal plasma. These platelets did not agglutinate spontaneously, or at lower ristocetin concentrations than platelets that were not treated with thrombin before formalin fixation. Platelets were also activated by thrombin in the presence of EDTA to prevent surface binding of VIII:vWF or secreted PF4, and then formalinized. These platelets did not bind VIII:vWF in the presence of purified PF4. We conclude that even though PF4 binds to both polyanions and the platelet membrane, it does not promote the attachment of VIII:vWF.

Identification of electrocorticogram patterns as the basis for a direct brain interface.

This study reports on the first step in the development of a direct brain interface based on the identification of event-related potentials (ERPs) from an electrocorticogram obtained from the surface of the cortex. Ten epilepsy surgery patients, undergoing monitoring with subdural electrode strips and grid arrays, participated in this study. Electrocorticograms were continuously recorded while subjects performed multiple repetitions for each of several motor actions. ERP templates were identified from action-triggered electrocorticogram averages using an amplitude criterion. At least one ERP template was identified for all 10 subjects and in 56% of all electrode-recording sets resulting from a subject performing an action. These results were obtained with electrodes placed solely for clinical purposes and not for research needs. Eighty-two percent of the identified ERPs began before the trigger, indicating the presence of premovement ERP components. The regions yielding the highest probability of valid ERP identification were the sensorimotor cortex (precentral and postcentral gyri) and anterior frontal lobe, although a number were recorded from other areas as well. The recording locations for multiple ERPs arising from the performance of a specific action were usually found on close-by electrodes. ERPs associated with different actions were occasionally identified from the same recording site but often had noticeably different characteristics. The results of this study support the use of ERPs recorded from the cortical surface as a basis for a direct brain interface.

Detection of event-related potentials for development of a direct brain interface.

The study presented here is part of an ongoing effort to develop a direct brain interface based on detection of event-related potentials (ERPs). In a study presented in a companion article, averaged ERP templates were identified from electrocorticograms recorded during repetition of voluntary motor actions. Here the authors report on the detection of individual motor ERPs within the electrocorticogram using cross-correlation. An averaged ERP template was created from the first half of each electrocorticogram and then cross-correlated with the continuous electrocorticogram from the second half. Points where the cross-correlation value exceeded an experimentally determined detection threshold were considered to be detection points. A detection point was considered to be a valid "hit" if it occurred between 1 second before and 0.25 second after the recorded time of a voluntary action. The difference between the hit and false-positive percentages (HF-difference) was used as a metric of detection accuracy. HF-differences greater than 90 were found for 5 of 15 subjects, HF-differences greater than 75 were found for 8 of 15 subjects, and HF-differences greater than 50 were found for 12 of 15 subjects. The three other subjects with HF-differences less than 50 had electrode locations not well suited for recording movement-related ERPs. Recordings from sensorimotor and supplementary motor areas produced the highest yield of channels with HF-difference greater than 50; however, a number of channels with good performance were found in other areas as well. The results demonstrate the likely prospect of using ERP detection as the basis of a single-switch direct brain interface and that furthermore, there is a good possibility of obtaining multiple control channels using this approach.

Case report: the lupus anticoagulant-hypoprothrombinemia syndrome.

The lupus anticoagulant is a well-described in vitro phenomenon that may be associated with arterial and venous thrombotic episodes. The lupus anticoagulant is never accompanied by a hemorrhagic diathesis unless it is associated with a second coagulation abnormality such as thrombocytopenia or hypoprothrombinemia. The lupus anticoagulant-hypoprothrombinemia syndrome is now a well-defined entity that may cause a severe, life-threatening hemorrhagic diathesis. The hypoprothrombinemia in this syndrome is the result of rapid clearance of prothrombin-antiprothrombin antibody complexes by the reticulo-endothelial system. The cause of antiprothrombin antibody formation is unknown. The authors describe a recent experience with a patient with this syndrome who initially had recurrent, life-threatening gastrointestinal bleeding. They were able to demonstrate hypoprothrombinemia and the presence of prothrombin-antiprothrombin antibody immune complexes. The patient was treated with prednisone, with correction of the bleeding disorder; however, the patient had resultant death from thrombosis. A literature review of the past 30 years as it relates to the discovery and treatment of this phenomenon is included.

Brain-computer communication based on the dynamics of brain oscillations.

This chapter presents a review of brain-computer communication based on motor imagery and the dynamics of brain oscillations. The concept of motor imagery as experimental strategy and the two different modes of operation a brain-computer interface can have are explained. An EEG based brain switch that can control a FES-induced hand grasp of a tetraplegic and an approach towards an ECoG based brain switch are presented.

Electrical muscle stimulation for pressure variation at the seating interface.

A new method is proposed for pressure sore prevention using electrical muscle stimulation (EMS). Potential mechanisms through which EMS may act for this purpose are discussed, including both short-term/dynamic and chronic effects. Measurements of maximum pressure variation in three able-bodied subjects using low levels of stimulation were performed. Pressure distribution changes were also measured. Fatigue effects on pressure redistribution were studied for four able-bodied subjects as well as for one C4, complete spinal cord injured individual. The results indicate that EMS produces sizeable pressure reduction under the ischial tuberosity, with redistribution occurring over other parts of the seating surface in able-bodied subjects. Fatigue effects were not observed in the four able-bodied subjects even after prolonged stimulation. Fatigue was observed with the spinal cord injured subject, but only after extensive stimulation. These studies demonstrate the feasibility of using EMS at relatively low intensity to vary seating interface pressure. The results warrant continued investigation of EMS to assist in pressure sore prevention.

Erythema and skin temperature following continuous sitting in spinal cord injured individuals.

Pressure sores are a severe and costly problem for many disabled individuals. There is a need for quantitative tools to assess damage produced by external loads on human skin and underlying tissues. Clinically, intensity and size of skin erythema have been used as indicators of tissue damage. Temperature is a quantifiable measure, and various studies have investigated the thermal response to localized pressure. The purpose of this study was to measure the effect of "long-term sitting" on skin temperature and erythema, in a situation that closely approximated what a spinal cord injured individual encounters on a regular basis. The resulting data indicated that: 1) a consistent skin temperature pattern occurred after pressure relief from the seated position; 2) skin temperature of experimentally-induced erythematous areas often remained elevated, even after one hour of pressure relief; and, 3) a qualitative, but not quantitative, correlation exists between erythema size and intensity and skin temperature. Implications of this research include the potential use of temperature to: 1) monitor the effectiveness of various strategies being used to prevent the development of pressure sores; and, 2) predict incipient tissue damage.

Evaluation of virtual source beam configurations for rapid tomographic reconstruction of gas and vapor concentrations in workplaces.

Beam path average data from an Open Path Fourier Transform Infrared (OP-FTIR) spectrometer can be used to reconstruct two-dimensional concentration maps of the gas and vapor contaminants in workplaces and the environment using computed tomographic (CT) techniques. However, a practical limitation arises because in the past, multiple-source and detector units were required to produce a sufficient number of intersecting beam paths in order to reconstruct concentration maps. Such a system can be applied to actual field monitoring situations only with great expense and difficulty. A single monostatic OP-FTIR system capable of rapid beam movement can eliminate this deficiency. Instead of many source and detector units, a virtual source arrangement has been proposed using a number of flat mirrors and retroreflectors to obtain intersecting folded beam paths. Three virtual source beam configurations generated for a single-beam steerable FTIR system were tested using 54 flat mirrors and four retroreflectors or 54 flat mirrors and 56 retroreflectors mounted along the perimeter walls of a typical 24- x 21-ft test room. The virtual source CT configurations were numerically evaluated using concentration maps created from tracer gas concentration distributions measured experimentally in a test chamber. Synthetic beam path integral data were calculated from the test maps and beam configurations. Computer simulations of different beam configurations were used to determine the effects of beam geometry. The effects of noise and peak reducing artifacts were evaluated. The performance of the tomographic reconstruction strategy was tested as a function of concentration and concentration gradients.

Tomographic reconstruction of tracer gas concentration profiles in a room with the use of a single OP-FTIR and two iterative algorithms: ART and PWLS.

Computed tomographic (CT) reconstructions of air contaminant concentration fields were conducted in a room-sized chamber employing a single open-path Fourier transform infrared (OP-FTIR) instrument and a combination of 52 flat mirrors and 4 retroreflectors. A total of 56 beam path data were repeatedly collected for around 1 hr while maintaining a stable concentration gradient. The plane of the room was divided into 195 pixels (13 x 15) for reconstruction. The algebraic reconstruction technique (ART) failed to reconstruct the original concentration gradient patterns for most cases. These poor results were caused by the "highly underdetermined condition" in which the number of unknown values (156 pixels) exceeds that of known data (56 path integral concentrations) in the experimental setting. A new CT algorithm, called the penalized weighted least-squares (PWLS), was applied to remedy this condition. The peak locations were correctly positioned in the PWLS-CT reconstructions. A notable feature of the PWLS-CT reconstructions was a significant reduction of highly irregular noise peaks found in the ART-CT reconstructions. However, the peak heights were slightly reduced in the PWLS-CT reconstructions due to the nature of the PWLS algorithm. PWLS could converge on the original concentration gradient even when a fairly high error was embedded into some experimentally measured path integral concentrations. It was also found in the simulation tests that the PWLS algorithm was very robust with respect to random errors in the path integral concentrations. This beam geometry and the use of a single OP-FTIR scanning system, in combination with the PWLS algorithm, is a system applicable to both environmental and industrial settings.

Evaluation of a nitrogen-cooled, electrically heated cold trap inlet for high-speed gas chromatography.

Gas chromatography has the potential to be a much faster method of separation than is usually realized. If column operating conditions are optimized for speed and injection band width is minimized, some simple separations can be completed in a few seconds. A prototype cryofocusing system for producing narrow injection bands with 0.25-mm i.d. columns is described here. The gas-cooled and electrically heated inlet produces injection bands with widths of about 10-20 ms. In the present study the system is evaluated using mixtures of common organics, including alkanes, aromatics, alcohols, ketones, and chlorinated hydrocarbons. Quantitative trapping and reinjection is achieved for all tested compounds. Coefficients of variation are less than 3% for peak area and less than 0.2% for retention time. Base-line separation of simple mixture is achieved with retention times of less than 10 s. By using the cold trap inlet with a low-dead-volume detector and a high-speed electrometer, the efficiency available from commercial capillary columns can be better utilized, and retention times for some routine separations may be reduced to a few seconds.

Learning and performance of able-bodied individuals using scanning systems with and without word prediction.

This study examines how the cognitive and perceptual loads introduced by a word prediction feature impact learning and performance. Two groups of able-bodied subjects transcribed text using two row-column scanning systems for 10 consecutive trials each. The two systems differed only in that one system had a word prediction feature. Subject groups differed in their order of system use. The results show that, under the conditions of this study, the word prediction system was not substantially more difficult to learn, but it did not yield a statistically significant improvement in text generation rate. This suggests that the cost of using this word prediction system balanced the benefit of the keystroke savings achieved by these subjects. The relationship between keystroke savings, cost in item selection rate, and improvement in text generation rate is explored in order to provide insight into this outcome.