Microtubule reassembly in surface-activated platelets

It is generally accepted that a circumferential microtubule supports the discoid shape of resting platelets. The fate of the many-coiled polymer following platelet activation, however, has been a subject of considerable debate. Morphological investigations have suggested that the circumferential coils are constricted into tight rings around centrally concentrated organelles during platelet shape change. Biochemical studies employing colchicine-binding assays, on the other hand, have indicated that the bundle of microtubules dissolves almost completely within seconds after activation and reassembles in a new location one to four minutes later. The present study has accepted the latter hypothesis in order to examine the second part of the disassembly-reassembly theory proposed in biochemical studies. Platelets exposed to low temperatures sufficient to remove all microtubules were placed on glass slides and microscope grids to cause surface activation during rewarming. The combined stimuli of rewarming and surface activation might have been expected to cause more rapid assembly than warming alone or activation alone. This was not the case. Reassembly of microtubules during rewarming and simultaneous surface activation was not accelerated. In contrast to the constriction of microtubule rings observed during activation in control platelets, the diameters of coils that developed in chilled platelets one to two hours after rewarming and surface activation were twice those of control cells.     

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.     

Zinc protoporphyrin in anemia of chronic disorders

Hematofluorometric determination of zinc protoporphyrin (ZPP) is a screening method for the assessment of iron deficiency (ID). Chronic disorders are frequently accompanied by anemias of unclear origin, most probably caused by an impairment of iron metabolism. We investigated the relevance of ZPP for the detection of derangements of iron metabolism in anemias of chronic disorders (ACD). In 19 patients with ACD caused by chronic inflammatory non-neoplastic diseases, ZPP was determined and correlated with ferritin, transferrin saturation, and hemoglobin (Hb). Marrow sideroblast counts and semiquantitative grading of the marrow hemosiderin were performed in all patients to exclude ID and to show the decreased iron bioavailability. In all ACD patients who exhibited the typical laboratory findings of disturbed iron metabolism, such as hypoferremia, decreased transferrin saturation, decreased bone marrow sideroblasts, and increased marrow hemosiderin, strongly elevated ZPP levels were found (131 +/- 23 mumol/mol heme). ZPP returned to normal after successful treatment of the underlying disease. This is shown in three patients with polymyalgia rheumatica. We conclude that the fluorometric determination of ZPP allows detection and quantification of derangements of iron metabolism associated with chronic inflammatory disorders. By recording the derangements quantitatively, ZPP allows monitoring of therapy of chronic inflammatory diseases.     

Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC. This decrease was dose dependent and could be prevented by specific anti-protein C antibodies. No decrease was observed with the zymogen protein C or with diisopropylfluorophosphate-inactivated APC. APC also decreased the t-PA inhibitor activity in endothelial cell-conditioned medium in the absence of cells, which suggests that the effect of APC is at least partly due to a direct effect of APC on the plasminogen activator- inhibitor. High concentrations of thrombin-but not of factor Xa or IXa-- had a similar effect on the t-PA inhibitor activity. The effect of APC on the plasminogen activator-inhibitor provides a new mechanism by which APC may enhance fibrinolysis. The data suggest that activation of the coagulation system may lead to a secondary increase of the fibrinolytic activity by changing the balance between plasminogen activator(s) and its (their) fast-acting inhibitor.     

Improving sensitivity of cervical cytology by removal of cervical secretions before sampling: a prospective study in Mexico

Sensitivity of cervical cytology is suboptimal, especially in developing countries such as Mexico, despite available guidelines aimed at improving this. When obtaining cervical samples, whether the samples are taken from the transformation zone and whether abnormal cells are missing must be considered. Cervical secretions (CS) are always present in variable proportions, and when cleaning the cervix, better samples may be obtained. In this study, we analyzed samples obtained with or without cleaning the cervix, and compared their contents in order to determine the sensitivity and specificity of these two methods. Methods: Of 500 patients who underwent cytology and colposcopy, 271 (54.2%) required a second opinion due to a diagnosis of cervical intraepithelial neoplasia (CIN). CS was removed and compared with the clean, second sample (SS) using in both liquid-based cytology. The quality of samples according to the Bethesda System, the presence of CIN, and inflammatory reactions were recorded. The sensitivity and specificity were calculated using biopsy as the gold standard. Results: The SS resulted in a higher proportion of adequate samples being obtained (97.6% vs. 44.8%), and in increased sensitivity (88.2% vs. 58.8%). CIN was detected in the SS 26% more often than in the CS (34 vs. 27 samples), whereas inflammatory reactions were noted more often in the CS (91.4% vs. 74%). Conclusion: Cervical sampling including CS results in lower sensitivity and CIN detection rates, and in more inflammatory reactions. By excluding CS from cervical samples, the sensitivity could be improved and the false negative rate could be reduced.