Neutrophil cathepsin G modulates the platelet surface expression of the glycoprotein (GP) Ib-IX complex by proteolysis of the von Willebrand factor binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex.

The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb-IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin-induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed against the macroglycopeptide portion of GPIb alpha). In contrast to its proteolytic effect on GPIb alpha, the cathepsin G-induced decrease in platelet surface GPIX and the remnant of the GPIb-IX complex (defined by MoAbs FMC25 and AK1) was via a cytoskeletal-mediated redistribution, as determined by lack of change in the total platelet content of GPIX and the GPIb-IX complex; complete inhibition by cytochalasin B, prostaglandin I2, and prior fixation of platelets. Experiments with Serratia protease-treated and Bernard-Soulier platelets showed that neither platelet surface GPIb nor cathepsin G-induced proteolysis of GPIb were required for the cathepsin G-induced redistribution of the remnant of the GPIb-IX complex or the cathepsin G-induced increase in platelet surface P-selectin. In summary, neutrophil cathepsin G modulates the platelet surface expression of the GPIb-IX complex both by proteolysis of the vWF binding site on GPIb alpha and by a cytoskeletal-mediated redistribution of the remainder of the complex. Prior studies show that, although thrombospondin 1, antiserine proteases, and plasma are all inhibitors of cathepsin G, the effects of cathepsin G on platelets, including an increase in surface GPIIb-IIIa, occur during close contact between neutrophils and platelets in a protective microenvironment (eg, thrombosis and local inflammation).(ABSTRACT TRUNCATED AT 400 WORDS)

Human neutrophil cathepsin G is a potent platelet activator.

PURPOSE: Neutrophil activation has been implicated in the pathophysiologic condition of ischemia-reperfusion injury, the formation of arterial aneurysms, the progression of myocardial ischemia, and the initiation of deep venous thrombosis. Activated neutrophils release cathepsin G, a serine protease, from their granules, which may cause platelet activation that leads to intravascular thrombosis, tissue infarction, and systemic release of the thrombogenic products of platelet granules. This study used flow cytometry to quantify the extent of cathepsin G-induced platelet activation and degranulation through changes in the expression of platelet surface glycoproteins. METHODS: Increasing concentrations of human neutrophil-derived cathepsin G were incubated with washed platelets or whole blood from healthy human donors. The platelet surface expression of glycoproteins, including P-selectin, a platelet membrane glycoprotein only expressed after platelet alpha granule release, were determined by quantifying the platelet binding of a panel of fluorescently labeled monoclonal antibodies. Results were compared with the effect of a maximal dose of thrombin, the most potent known platelet activator. RESULTS: In a washed platelet system, cathepsin G increased platelet surface expression of P-selectin (an activation-dependent neutrophil binding site), the glycoprotein IIb/IIIa complex (fibrinogen receptor), and glycoprotein IV (thrombospondin receptor), and decreased surface expression of glycoprotein Ib (von Willebrand factor receptor) to an extent comparable to maximal thrombin. However, these effects were not observed in a whole blood system. Further experiments revealed that preexposure to plasma completely inhibited cathepsin G-induced washed platelet activation and degranulation. Prostacyclin treatment of washed platelets markedly inhibited cathepsin G-induced platelet activation. CONCLUSIONS: Cathepsin G is a very potent platelet agonist and degranulator, comparable to maximal thrombin, which alters platelet surface glycoprotein expression for enhanced neutrophil binding and effective platelet aggregation. This study helps to elucidate a possible pathway through which neutrophils may directly activate platelets, leading to intravascular thrombosis, irreversible ischemia, and tissue death in cardiovascular disease states. Patients with diseased endothelium that is deficient in prostacyclin production may be particularly prone to the detrimental effects of neutrophil-derived cathepsin G platelet activation.

Parathyroid localization prior to primary exploration.

The success of parathyroid surgery is based on accurate localization of normal and abnormal parathyroid glands, knowledge of the pathologic conditions, and meticulous dissection during removal of the abnormal glands. Although parathyroid localization is essential in cases requiring re-exploration, there is considerable controversy regarding the indications for localization studies prior to primary exploration, since the success rate for surgery exceeds 90% to 95%. However, in specific circumstances, including diagnostic problems, technical considerations, and high-risk patient factors, preoperative parathyroid localization assists the operating surgeon even during the primary cervical exploration. The purpose of this paper is to define these specific circumstances and discuss the appropriate studies.

The adaptive ability of transplanted rat small intestine.

This study was performed to determine the extent to which intestinal transplants undergo functional and morphologic compensation. Animals were studied after 25 days to document how rapidly the changes occurred and after 150 days to establish whether the effects were maintained long term. Lewis isografts and Lewis Brown Norway F1 allograft recipient rats had comparable degrees of morphologic (increased bowel diameter, crypt depth, and villus height) and functional (absorption of 3H-glucose, 14C-maltose, and cyclosporine) compensation. These changes were already present by day 25 and persisted until at least day 150. The results were independent of the loss of extrinsic innervation or the intramuscular administration of cyclosporine (5 mg/kg/day). These observations support the usefulness of segmental intestinal transplantation in the treatment of surgically induced short bowel syndrome.

Successful segmental intestinal transplantation in enterectomized pigs.

The aim of this study was to determine whether short-segment jejunal allografts maintained the viability and nutritional status of outbred recipient pigs treated with low-dose cyclosporine. The animals were subjected to total small bowel resection (from the ligament of Treitz to the ileocecal valve, approximately 15 m). Short-gut control animals (n = 8) who had no transplant died of malabsorption on day 62.5 +/- 4.1 (mean +/- SEM). Without cyclosporine immunosuppression, recipients (n = 5) of 3 m to 4 m jejunal allografts died of rejection on day 8.8 +/- 0.7. However enterectomized pigs (n = 11) who had segmental jejunal allograft transplants and were treated with cyclosporine (10 mg/kg/day) demonstrated significantly prolonged survival (to day 80.9 +/- 22.3; p less than 0.05). By 180 days after transplant, surviving animals increased their weight by almost 40%. In conclusion short-segment jejunal allografts significantly improved the mortality and morbidity rates from surgically created short bowel syndrome in pigs.

Effect of transplantation on tissue levels of substance P, vasoactive intestinal polypeptide, and serotonin in rat small intestine.

Denervation of the gut, resulting in altered bowel function, has been viewed as an impediment to the clinical success of small intestinal transplantation. This study examined the effect of complete extrinsic denervation of the jejunum and ileum on tissue levels of VIP, SP, and 5-HT in a rat model of small intestinal transplantation. Orthotopic total small bowel isograft transplants were performed in 18 Lewis inbred rats. Sham operations consisted of occluding the superior mesenteric artery of 18 Lewis rats for 10 minutes to provide comparable degrees of ischemia. Six rats from each group were sacrificed 1, 2, and 4 weeks following transplantation or sham operation. The jejunum and ileum were removed and extracted in acid for measurement of VIP, SP, and 5-HT by radioimmunoassay. There were no statistically significant differences in the jejunal or ileal content of VIP or 5-HT or the jejunal content of SP between the transplant and sham groups. An initial decrease in ileal SP content at 1 week following transplantation was no longer evident by the fourth week. We conclude that the extrinsic denervation of small intestinal transplantation has minimal effects on the intestinal content of VIP, SP, and 5-HT and should not significantly affect physiologic function controlled by these gastrointestinal hormones.

The role of serotonin in the canine secretory response to cholera toxin in vivo.

This study was initiated to evaluate the role of serotonin in cholera toxin-induced jejunal secretion of water and electrolytes. Chronic Thiry-Vella loops, constructed in six dogs, were perfused with an isosmotic neutral perfusate containing [14C]polyethylene glycol as the recovery marker. Fluxes of water, sodium, chloride and potassium were calculated and immunoreactive serotonin levels were measured in blood and effluent perfusates. Intraluminal application of 20 micrograms of cholera toxin induced secretion; fluxes of water (basal, 32.3 +/- 11.1; 6 hr, -541 +/- 35 microliter/min), sodium (basal, 9.0 +/- 2.8; 6 hr, -78.3 +/- 5.6 microEq/min), chloride (basal, 3.8 +/- 1.5; 6 hr, -65.7 +/- 4.0 muEq/min) and potassium (basal, 0.10 +/- 0.08; 6 hr, -2.80 +/- 0.18 muEq/min) were all significantly different from basal. Serum electrolytes remained normal, except that potassium fell from 4.9 +/- 0.5 to 3.9 +/- 0.2 mEq/l. Although circulating serotonin levels did not change from base line (180.9 +/- 29.3 ng/ml), effluent concentrations increased significantly from 68.2 +/- 4.6 to 81.1 +/- 5.0 ng/ml (at 3 hr) and jejunal outputs increased from 136.6 +/- 10.2 to 205.1 +/- 10.1 ng/min (at 6 hr). In a separate set of experiments, verapamil was infused i.v. (12.5 micrograms/kg/min) during the 4th hr in four dogs exposed to cholera toxin. The lower dose of toxin (5 micrograms) induced secretion which was unaffected by the calcium channel blocker. In another series of studies, ketanserin (a 5-HT2 receptor blocker) was infused i.v. at 33 micrograms/kg/min during the 4th hr in four additional dogs exposed to the lower dose of cholera toxin. This potent serotonin antagonist failed to inhibit cholera toxin-induced jejunal secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of small intestinal transplantation on intraluminal levels of serotonin and substance P.

This study was performed to examine the effect of transplantation, and thus extrinsic denervation, of the small intestine on intraluminal release of serotonin and substance P. Heterotopic 40-cm-long proximal (jejunal) small intestinal isografts were performed in six 200- to 250-g adult male Lewis rats under general anesthesia. Bowel ends were exteriorized as ostomies. Six Lewis rats with neurovascularly intact 40-cm proximal small bowel Thiry-Vella loops exteriorized as ostomies served as the control animals. On the seventh postoperative day, the intestinal loops were perfused at 0.5 ml/min for three 10-min periods with normal saline followed by an equilibrium period and then for three 10-min periods with 20% dextrose. Perfusates were collected for each period and levels of serotonin and substance P were determined by radioimmunoassay. Intraluminal serotonin levels rose from 29 +/- 9 ng/ml during saline perfusion to 115 +/- 28 ng/ml during intestinal perfusion with 20% dextrose in the innervated loops and from 21 +/- 7 ng/ml to 94 +/- 26 ng/ml in the transplanted loops. While there was a statistically significant increase in mean intraluminal serotonin levels following perfusion with 20% dextrose in both the control and transplant groups, there was no difference in the intraluminal serotonin response between controls and transplant recipients. In contrast, 20% dextrose had no effect on luminal release of substance P in either group. These results indicate that extrinsic denervation of the small intestine has no effect on the intraluminal serotonin response to stimulation and suggest that serotonin and substance P are not released into the intestinal lumen by the same regulatory mechanisms.

Prostaglandins and surgical diseases: Part II.

Prostaglandins are important in the allograft response. Allograft antigenic stimulation causes release of prostaglandins both in vivo and in vitro. Macrophages and monocytes are the cells that produce the prostaglandins from arachidonic acid via the cyclooxygenase pathway. Prostaglandins exert immunosuppressive effects at several steps on both afferent and efferent phases in the cell-mediated immune system: reduction of Ia expression in antigen-presenting cells, inhibition of IL-1 and IL-2 production, and activation of suppressor cells. The immunosuppressive effects of blood transfusions and essential fatty acids appear to be mediated by PGE.

Segmental intestinal transplantation in rats with resected entire small bowel, ileocecal valve, and cecum.

Segmental intestinal transplantation was studied in a rat model of severe short gut syndrome across major histoincompatibility barriers. Lewis (RT1l) recipient rats whose entire small bowel (approximately 80 cm), ileocecal valve, and cecum were resected and who had no transplant, uniformly died of malabsorption on Day 9.8 +/- 0.4 (n = 11). Without cyclosporine, allograft recipients (n = 2), died of rejection on Days 8 and 10. Recipient animals with 20-cm jejunum and 40-cm jejunal transplants from Buffalo (RT1b) rats and treated daily with cyclosporine (5 mg/kg/day) intramuscularly (Days 0-28) and vitamin B12 (every other week) enjoyed significantly prolonged survival to Day 58.2 +/- 13.7, P less than 0.003, n = 10, and Day 129.1 +/- 7.4, P less than 0.001, n = 10, respectively. While 7 of 10 rats in the 20-cm jejunal transplant group died of malabsorption between Days 14 and 58, none of 10 animals in the 40-cm jejunal transplant group died of this complication. Four of 10 rats in the 40-cm jejunal transplant group thrived at 150 days after the operation, at which time they were sacrificed. Morphologically, the grafts demonstrated hypertrophic changes. The data from this study suggest that intestinal allografts have pronounced intestinal adaptative characteristics. Using segmental jejunal grafts, intestinal transplantation is an effective surgical modality for the short gut syndrome in the rat.