We studied the adhesion of erythrocytes from 30 diabetic patients and 25 controls to human endothelial cells. Washed erythrocytes were labeled with 51Cr and added to confluent endothelial cells cultured from umbilical veins. After incubation at 37 degrees C, the nonadherent erythrocytes were removed by sequential washings. The percentage of erythrocytes adhering to cultured endothelium after each wash was significantly higher when erythrocytes were from diabetics than when they were from controls (P less than 0.005). After the fifth wash, the mean adhesion ratio (percentage of adhering diabetic red cells: percentage of adhering control red cells) was 2.33 (range, 0.8 to 5.2). Increased adhesion was related to the extent of vascular complications in the diabetics, as assessed by a vascular score. With the same technique, fewer erythrocytes adhered to plastic and to cultured human fibroblasts than to endothelial cells, although the adhesion of the diabetic red cells to these surfaces was higher than that of the controls. These results suggest that in diabetes there is an intrinsic erythrocyte abnormality that is related to vascular disease.
Painful vasocclusive episodes are one of the most prominent pathological features of sickle cell disease. In addition to abnormal shape and poor deformability, increased adhesion of red cells to endothelium has been reported. On several occasions, we have studied the adhesion of erythrocytes from 30 patients with mixed sickle cell syndromes to evaluate the influence of clinical conditions. The percentage of erythrocytes adhering was significantly higher when erythrocytes from sickle patients were compared with controls (p less than 0.01). Furthermore, adhesion was significantly higher when the patients were in crises (p less than 0.01), and the highest values of all were observed in patients with inflammatory conditions. To investigate the possibility that a limited population of red cells could be responsible for the increase in red cell adhesion, we have measured the HbS concentration in the different washes and found that the HbS concentration was higher in the last washes compared to the first washes. Sickle red cells capable of protein synthesis (young red cells) were labelled with [3H] leucine. The adhesion to endothelial cells of [3H] leucine-labelled red cells was higher than that of the 51Cr-labelled red cells from the same patient. On the other hand, the most dense sickle red cells separated by density gradient adhered to a greater extent than the light red cells. This apparent discrepancy could be partly explained by the presence of [3H] leucine-labelled red cells in the dense fractions of sickle red cells separated by stractan gradient.
Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human cr-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x mol . 1-l was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 pM and a K , of 1.72 pM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 f 0.12 and an apparent dissociation constant of 57.0 13.1 pM (mean f SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.Aprotinin [l], a well-known basic proteinase inhibitor isolated from bovine tissues, is of particular interest because of its broad inhibitory specificity. It is a reversible and competitive inhibitor of various proteinases, the active site of the enzyme being blocked by an equimolar complex formation. Besides trypsin and chymotrypsin, it strongly inhibits plasmin, kallikrein and protein C [l, 21. This specificity accounts for its initial therapeutic use, particularly in pancreatisis, various states of shock or hyperfibrinolytic hemorrhage [I]. More recently, the administration of aprotinin during cardiac surgery has been shown to reduce the peroperative and postoperative bleeding, leading to a reduction in the transfusion requirement [3 -61.The beneficial effect of aprotinin on hemostasis was ascribed to protection of the plasma proteins cascade from activation and to platelet preservation [6 -81. Platelet preservation can be ensured by the protection of platelet receptors from exposure and/or degradation by agonists ...
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