Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.     

Prospective use of platelet glycoprotein IIb/IIIa receptor blockers in the emergency department setting

Platelets play a pivotal role in the pathophysiology of acute coronary syndromes (ACS) and thus are logical therapeutic targets for treatment of this disease process. Platelet glycoprotein (GP IIb/IIIa receptor antagonists, which interrupt the final common pathway of platelet aggregation, have been proven to reduce the 30-day incidence of death, acute myocardial infarction (MI), and urgent revascularization in both high-risk and low-risk patients undergoing percutaneous intervention procedures. Three-year follow-up has indicated that these benefits appear durable. Recent large-scale randomized trials have demonstrated the value of GP IIb/IIIa receptor inhibitors in reducing the risk of death and MI in unstable angina/non-Q-wave MI patients receiving pharmacologic management. And, emerging evidence suggests a future role for GP IIb/IIIa receptor inhibitors as an adjunct to low-dose fibrinolytic therapy in patients with acute MI. As the list of indications for GP IIb/IIIa receptor antagonists expands to encompass the full spectrum of ACS, there is increasing interest in the potential use of these agents in the emergency department setting. The integration of GP IIb/IIIa receptor inhibitors into emergency department protocols will ultimately depend largely on whether these drugs prove to be safe and effective regardless of the direction of ST-segment deviation, and irrespective of whether definitive therapy will be invasive or conservative.     

Individualized assessment and intervention in bilateral siderail use

The use of bilateral siderails, similar to physical restraints, can be safely reduced by a comprehensive assessment process. This article presents an individualized assessment for evaluating siderail use to guide nurses in managing resident characteristics for falling out of bed and intervening for high-risk residents. The individualized assessment is consistent with federal resident assessment instrument requirements and includes risk factors specific to falls from bed.     

Effect of visual cues, vital signs, and protocols on triage: a prospective randomized crossover trial

The purpose of this study is to review published data regarding gender differences in cardiac electrophysiology and in the occurrence of clinical arrhythmias. ECG differences between men and women include a faster resting heart rate in women, a longer corrected QT interval, and a lower QT dispersion than in men. The faster resting heart rate in women appears to be primarily related to differences in physical conditioning. The mechanism for the longer corrected QT interval in women is not completely known, but does not appear to be related to acute effects of estrogen or progesterone or differences in autonomic innervation. Women also appear to have a lower incidence of atrial fibrillation, a difference in the age distribution of supraventricular tachycardia, and a lower incidence of sudden death than men. Much of the lower incidence of sudden death in women may relate to a difference in the prevalence of coronary artery disease, but other factors such as inherent differences in repolarization, which may be reflected by a gender difference in the corrected QT interval, also may be operative. The paradox of a longer corrected QT interval and higher incidence of torsades de pointes, but lower population-based incidence of sudden death in women, has not been completely resolved. Further studies will be required to help better understand the basic mechanisms involved in gender differences in electrophysiology and arrhythmias and determine the extent to which these differences have implications for clinical management of cardiac arrhythmias.     

Conditioning heat stress reduces excitotoxic and apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro

Both accidental and experimental lesions of the spinal cord suggest that neuronal processes occurring in the spinal cord modify the relay of information through the dorsal column-lemniscal pathway. How such interactions might occur has not been adequately explained. To address this issue, the receptive fields of mechanosensory neurons of the dorsal column nuclei were studied before and after manipulation of the spinal dorsal horn. After either a cervical or lumbar laminectomy and exposure of the dorsal column nuclei in anesthetized cats, the representation of the hindlimb or of the forelimb was defined by multiunit recordings in both the dorsal column nuclei and in the ipsilateral spinal cord. Next, a single cell was isolated in the dorsal column nuclei, and its receptive field carefully defined. Each cell could be activated by light mechanical stimuli from a well-defined cutaneous receptive field. Generally the adequate stimulus was movement of a few hairs or rapid skin indentation. Subsequently a pipette containing either lidocaine or cobalt chloride was lowered into the ipsilateral dorsal horn at the site in the somatosensory representation in the spinal cord corresponding to the receptive field of the neuron isolated in the dorsal column nuclei. Injection of several hundred nanoliters of either lidocaine or cobalt chloride into the dorsal horn produced an enlargement of the receptive field of the neuron being studied in the dorsal column nuclei. The experiment was repeated 16 times, and receptive field enlargements of 147-563% were observed in 15 cases. These data suggest that the dorsal horn exerts a tonic inhibitory control on the mechanosensory signals relayed through the dorsal column-lemniscal pathway. Because published data from other laboratories have shown that receptive field size is controlled by signals arising from the skin, we infer that the control of neuronal excitability, receptive field size and location for lemniscal neurons is determined by tonic afferent activity that is relayed through a synapse in the dorsal horn. This influence of dorsal horn neurons on the relay of mechanosensory information through the lemniscal pathways must modify our traditional views concerning the relative independence of these two systems.     

Conventional color Doppler velocity sonography versus color Doppler energy sonography for the diagnosis of acute experimental torsion of the spermatic cord

OBJECTIVE: We compared color Doppler velocity sonography and color Doppler energy sonography for the diagnosis of spermatic cord torsion in a canine model and determined the degree of torsion necessary to acutely halt testicular blood flow. MATERIALS AND METHODS: Spermatic cord torsion was created in five dogs by exposing and rotating the ipsilateral testis 0 degree, 180 degrees, 270 degrees, 360 degrees, 450 degrees, and 540 degrees. Detorsion followed. The testicles were scanned at each torsion stop using both color Doppler velocity sonography and color Doppler energy sonography. Doppler parameters were optimized (by phantom and test scans) and maintained at a tolerable noise level throughout the experiment. Readers who were unaware of the degree of torsion compared flow in the rotated and contralateral control testes. RESULTS: Flow became undetectable by color Doppler velocity sonography and color Doppler energy sonography at 450 degrees in four of five cases and at 540 degrees in one of five cases. We found no significant difference between the velocity and the energy techniques for detecting this absence of flow (p > .05, Wilcoxon test). We found a significant difference in degree of flow for both techniques when comparing controls and all degrees of torsion combined (p < .006, Mann-Whitney test), but significance was achieved at lesser degrees of torsion with the velocity technique than with the energy technique (180 degrees and 360 degrees, respectively, Wilcoxon test). CONCLUSION: Color Doppler energy sonography was not significantly more sensitive than color Doppler velocity sonography for the diagnosis of spermatic cord torsion in this model. Complete occlusion of arterial inflow occurred at 450-540 degrees of torsion.     

No increased incidence of Alzheimer's disease in elderly schizophrenics

There is currently controversy as to the morphological basis of cognitive impairment in elderly schizophrenics. In contrast to previous findings, recent studies have found no increased frequency of Alzheimer's disease (AD) pathology in elderly schizophrenics. We examined 99 consecutive autopsy cases of patients over the age of 55 years from a psychiatric hospital who met the DSM-III-R and ICD.10 criteria for schizophrenia (mean age 69.5 +/- 8.25 years; mean duration of illness 35.15 +/- 10.1 years), 56% showing moderate to severe dementia. All brains were blindly reviewed for evidence of AD using CERAD criteria and Braak staging of neuritic AD lesions. "Definite" AD (CERAD C, Braak stage V) was seen in 2 cases aged 56 and 67 years, respectively [2% of total or 1/68 (1.4%) of those over age 65]. "Probable" AD (CERAD B, Braak stages IV-V) were seen in 5 cases aged 71-89 years (mean 79 years; 5% of total or 7.3% of those over age 65), and 1 case each with multiple cerebral infarcts and with Parkinson's disease pathology. In addition, 2 females aged 82 and 89 years, respectively, revealed senile dementia with tangles (NIA, CERAD negative; Braak stage IV), 1 with hippocampal sclerosis. The total incidence of definite and probable AD in this cohort was 7.1% or 8.7% for those over age 65. This is in line with other recent studies showing that the frequency of AD in elderly schizophrenics may be equal or even less than in the general population. The reasons for this negative association and the basis of cognitive deficits in elderly schizophrenics--those with dementia usually showing significantly lower brain weight--await further elucidation.     

Cooperative interactions between adjacent troponin-tropomyosin complexes may be transmitted through the actin filament

Recent analyses of the assembly of thin filaments containing altered forms of troponin (or no troponin) suggested that the strongly cooperative nature of troponin-tropomyosin binding to actin might be primarily caused by indirect interactions involving the actin lattice, rather than by direct contacts between neighboring troponin-tropomyosin molecules. To test this hypothesis, thin filament assembly was examined using either cardiac tropomyosin digested with carboxypeptidase A (cbpTm) or a tropomyosin with defective function at both amino and carboxyl termini (unacetylated cbpTm). Compared to intact troponin-tropomyosin, both troponin-cbpTm and troponin-unacetylated cbpTm had much weaker binding to actin; however, cooperative interactions were only slightly reduced. These data support the implication that the primary source of the cooperativity involves troponin-tropomyosin-promoted conformational changes within the actin polymer. Surprisingly, the effects of tropomyosin amino- and carboxyl-terminal structural defects on troponin-tropomyosin binding to actin were not additive. In the presence of troponin, tropomyosin molecules with either defect had the same diminution in actin affinity as molecules with both defects. Finally, the Ca2+ sensitivity of troponin-tropomyosin binding to actin was increased by alteration of either end of tropomyosin.     

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.     

Differential proliferative and interleukin-10 responses to fractionated filarial antigens: preferential recognition by patients with chronic lymphatic dysfunction

To characterize filarial antigens that may be associated with the development of chronic lymphatic dysfunction in persons with lymphatic filariasis, T cell responsiveness to Brugia pahangi adult worm extracts and SDS-PAGE antigen fractions were examined among Haitians from an area in which Wuchereria bancrofti is endemic. Greater T cell proliferation and interleukin-10 (IL-10) production were observed in amicrofilaremic patients with hydrocele or elephantiasis than in amicrofilaremic or microfilaremic asymptomatic persons. Antigen fractions that stimulated the highest proliferative responses (in the 25-49 kDa range) and IL-10 production were not identical. Further separation of an immunodominant 30- to 38-kDa fraction by ion exchange high-pressure liquid chromatography identified several subfractions, including a 32-kDa protein band, that elicited T cell responses from patients with elephantiasis or hydrocele. By immunoblot, these patients also had markedly greater humoral reactivity to parasite antigens of approximately 52, 43, 32, and 30 kDa.