Platelet P-selectin and GPIIb/IIIa expression after liver transplantation and resection

Platelet dysfunction contributes to haemostatic defects, possibly leading to bleeding complications. We hypothesised that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. Therefore, we determined the expression of platelet GPIIb/IIIa and P-selectin, representing important platelet activation markers, and the thrombopoietin (TPO) serum level after transplantation and resection. Twenty patients [ten that had undergone orthotopic liver transplantation (OLT), ten with liver resection (LRX)] were included in the study. From sequential venous blood samples, surface expression of GPIIb/IIIa and P-selectin was quantified by flow cytometry, and TPO serum levels were determined by ELISA. Baseline GPIIb/IIIa receptor expression on circulating platelets was significantly reduced in the OLT group compared to the LRX group and healthy volunteers. GPIIb/IIIa expression after activation with TRAP-6 increased significantly (P<0.001) in the LRX group but not in the OLT group. P-selectin expression after TRAP-6 stimulation increased significantly (P<0.001) in the LRX group, being comparable to that in healthy volunteers, whereas only a very low increase in the OLT group was found. In the OLT group, TPO serum levels were in the lower normal range and rose above the upper limit of normal values 24 h after reperfusion. These data indicate that neither liver transplantation nor liver resection influences GPIIb/IIIa and P-selectin expression on circulating platelets. There was a lack of expression in cirrhotic patients and unimpaired baseline expression and functional reserve in non-cirrhotic liver-resection patients. After liver transplantation, increasing serum TPO levels, which indicated a recovering graft function, resulted in rising peripheral platelet counts.     

Differential platelet receptor expression following hydroxyethyl starch infusion in thrombocytopaenic orthotopic liver transplantation recipients

BACKGROUND AND OBJECTIVE: Platelet function abnormalities influence the haemostatic defect in patients with liver failure. Patients after orthotopic liver transplantation present thrombocytopaenia associated with bleeding problems, which may be aggravated by the interaction of hydroxyethyl starches with platelets. METHODS: From 12 patients after liver transplantation venous blood samples (3 mL) were taken before, 20 and 120 min after infusion of hydroxyethyl starch of medium molecular weight (200 kDa/0.5) 6% 10 mL kg(-1) over a period of 30 min. Surface expression of glycoprotein IIb/IIIa and P-selectin were quantified by flow cytometry as well as the percentage of platelet-leucocyte complexes. RESULTS: A significant decrease of P-selectin expression following administration of hydroxyethyl starch after 120 min (89.1 +/- 4.2%, P = 0.029) and a corresponding significant reduction in the formation of platelet-monocyte complexes (81.1 +/- 7.8%, P = 0.001) were observed. There was no alteration in the glycoprotein IIb/IIIa expression after hydroxyethyl starch infusion. CONCLUSIONS: Infusion of hydroxyethyl starch 200 kDa/0.5 in clinically relevant doses does not alter glycoprotein IIb/IIIa expression in thrombocytopaenic patients with pre-existing platelet dysfunction after orthotopic liver transplantation. Accordingly, infusion of hydroxyethyl starch may have a beneficial effect on microvascular graft perfusion through the resulting haemodilution and reduced P-selectin expression with subsequent reduced leucocyte-platelet complexes and endothelial adhesion.     

Sevoflurane inhibits unstimulated and agonist-induced platelet antigen expression and platelet function in whole blood in vitro.

BACKGROUND: Previous studies have reported conflicting results about the effect of sevoflurane on platelet aggregation. To clarify this point, we investigated the effects of sevoflurane on platelet antigen expression and function in vitro. METHODS: Human whole blood was incubated for 1 h with 0.5 and 1 minimum alveolar concentration sevoflurane, 21% O(2), and 5% CO(2). A control sample was kept at the same conditions without sevoflurane. After stimulation with adenosine diphosphate or thrombin receptor agonist peptide 6, samples were stained with fluorochrome conjugated antibodies, and the expression of platelet glycoproteins GPIIb/IIIa, GPIb, and P-selectin, as well as activated GPIIb/IIIa, were measured with two-color flow cytometry. In addition, platelet function was assessed by means of thromboelastography and using the platelet function analyzer 100. RESULTS: Already in subanesthetic concentrations, sevoflurane inhibits unstimulated and agonist-induced GPIIb/IIIa surface expression and activated GPIIb/IIIa expression on platelets in whole blood. The agonist-induced redistribution of GPIb into the open canalicular system was also impaired by sevoflurane, whereas no effect on P-selectin expression in activated platelets could be found. Sevoflurane significantly reduced the maximum thromboelastographic amplitude. Furthermore, platelet function analyzer 100 closure times were significantly prolonged. CONCLUSION: The results show that sevoflurane significantly impairs platelet antigen expression in vitro. It is especially the inhibition of GPIIb/IIIa expression and activation that impairs bleeding time as reflected in thromboelastographic measurements and platelet function analyzer 100 closure times. The exact inhibitory mechanism remains unclear.     

Sevoflurane Inhibits Unstimulated and Agonist-induced Platelet Antigen Expression and Platelet Function in Whole Blood In Vitro

Background : Previous studies have reported conflicting results about the effect of sevoflurane on platelet aggregation. To clarify this point, we investigated the effects of sevoflurane on platelet antigen expression and function in vitro.

Methods : Human whole blood was incubated for 1 h with 0.5 and 1 minimum alveolar concentration sevoflurane, 21% O2, and 5% CO2. A control sample was kept at the same conditions without sevoflurane. After stimulation with adenosine diphosphate or thrombin receptor agonist peptide 6, samples were stained with fluorochrome conjugated antibodies, and the expression of platelet glycoproteins GPIIb/IIIa, GPIb, and P-selectin, as well as activated GPIIb/IIIa, were measured with two-color flow cytometry. In addition, platelet function was assessed by means of thromboelastography and using the platelet function analyzer 100.

Results : Already in subanesthetic concentrations, sevoflurane inhibits unstimulated and agonist-induced GPIIb/IIIa surface expression and activated GPIIb/IIIa expression on platelets in whole blood. The agonist-induced redistribution of GPIb into the open canalicular system was also impaired by sevoflurane, whereas no effect on P-selectin expression in activated platelets could be found. Sevoflurane significantly reduced the maximum thromboelastographic amplitude. Furthermore, platelet function analyzer 100 closure times were significantly prolonged.     

A radical scavenger, edaravone, protects canine kidneys from ischemia-reperfusion injury after 72 hours of cold preservation and autotransplantation

BACKGROUND: Cold ischemia-reperfusion (I/R) injury is a prominent cause of delayed graft function after kidney transplantation. Reactive oxygen species play a crucial role in I/R injury. Edaravone is a synthetic radical scavenger that has been used in acute stroke. Some animal experiments have revealed its beneficial effects against I/R injury, but its effects after cold preservation and transplantation of canine kidneys are unknown. METHODS: Female hybrid dogs weighing 11 to 13 kg were used. Under anesthesia, the left kidney was harvested. After 72 hr of preservation in cold histidine-tryptophan-ketoglutarate solution, autotransplantation was performed in the right iliac fossa, with contralateral nephrectomy. Animals were divided into control and treatment groups (n=6 per group). In the treatment group, edaravone was administered intravenously at harvest and at reperfusion (3 mg/kg) and in addition was added to the preservation solution (50 microM). RESULTS: Animal survival at 2 weeks was four of six in the control group and six of six in the treatment group. Compared with controls, treated animals had higher mean urine output, higher mean glomerular filtration rate, improved tubular cell function, lower mean serum creatinine, and lower renal vascular resistance. Biopsy specimens from treated animals showed less tubular cell damage and decreased P-selectin expression in endothelial cells. Lipid peroxidation of renal tissue and urinary excretion of 8-hydroxy-2'-deoxyguanosine were suppressed by the treatment. CONCLUSIONS: Edaravone reduced cold I/R injury in canine renal transplantation. The agent has the potential to ameliorate preservation injury in clinical transplantation.     

Cold preservation mediated renal injury: involvement of mitochondrial oxidative stress

Cold preservation has greatly facilitated the use of cadaveric kidneys for renal transplantation, but, clearly, damage occurs during both the preservation episode and the reperfusion phase (following transplantation). The aims of this study were twofold: to develop an in vivo model that was capable of evaluating renal function at early time points following cold preservation, and to evaluate the extent of renal mitochondrial damage that occurs following short periods of cold preservation in vivo. To accomplish these goals, we developed a novel rat model of in vivo renal cold ischemia followed by warm reperfusion (cold I/R) which avoided the complexity involved with transplantation. Briefly, after a right nephrectomy, cold I/R was initiated via pulsatile perfusion (40 minutes) of the left kidney with a cold University of Wisconsin solution followed by 18 hours of warm reperfusion. Cold I/R resulted in significant renal injury, nitrotyrosine production, and inactivation of the key mitochondrial antioxidant enzyme, manganese superoxide dismutase. Furthermore, the activities of the mitochondrial respiratory complexes were significantly reduced following cold I/R. In conclusion, short-term cold I/R results in inactivation of MnSOD, which may lead to the inhibition of mitochondrial complexes and subsequent renal injury. These data suggest that compounds designed to prevent early mitochondrial injury in kidneys that undergo cold preservation would significantly improve renal function and graft survival following transplantation.     

Protection against ischemia/reperfusion injury by the cavitary two-layer method in canine small intestinal transplantation with reduction of reactive oxygen species

BACKGROUND: Ischemia and reperfusion (I/R) injury is a major determinant of early graft dysfunction and long-term graft survival in small intestinal transplantation. The cavitary two-layer method (TLM) has been reported to be superior to the University of Wisconsin cold storage method (UWM) in long-term preservation of canine small intestine. This study was designed to evaluate the protective effect of the cavitary TLM against I/R injury in canine small intestinal transplantation. METHODS: Intestinal grafts harvested from beagles were allotransplanted after 24-hour preservation by UWM (group 1) or the cavitary TLM (group 2). The graft in the controls (group 3) was immediately allotransplanted without preservation. I/R injury was assessed by functional success rates, biochemical assay, graft adenosine triphosphate (ATP) and lipid peroxidation (LPO) concentrations, and histopathologic examination including TUNEL staining for apoptosis. RESULTS: In group 1, ATP recovery was delayed after reperfusion, and most recipients died with hemorrhage of the grafts and lungs. In group 2, graft ATP concentrations recovered rapidly, and I/R injury was prevented with reduced LPO production, resulting in good outcome. CONCLUSIONS: The cavitary TLM protected intestinal grafts against I/R injury evidenced by maintenance of graft ATP levels and reduction of LPO production compared with UWM in canine small intestinal transplantation.     

Platelet glycoprotein IIb/IIIa receptor antagonist (abciximab) inhibited platelet activation and promoted skin flap survival after ischemia/reperfusion injury

BACKGROUND: Evidence has shown that platelets play an important role in the pathogenesis of flap failure. Employing a rat inferior epigastric artery skin flap as a flap reperfusion injury model, we investigated whether platelet activation was involved in the skin flap failure and whether administration of abciximab (ReoPro, chimeric 7E3 Fab) could decrease platelet activation/aggregation and promote flap survival. METHODS: Normal saline and abciximab (0.06 mg/kg; 0.2 mg/kg; 1 mg/kg) were injected intravenously into skin flaps 30 min before reperfusion and 1 h after reperfusion (each subgroup n = 6). Platelet activation as demonstrated by P-selectin (CD62P) was analyzed by flow cytometry. P-selectin expression on flap vessels was detected by immunohistochemical staining. Platelet aggregation was induced with adenosine diphosphate (ADP). Laser Doppler flowmetry monitored tissue perfusion. The surviving area was evaluated 7 days postoperatively. RESULTS: CD62P progressively increased after reperfusion. The peak CD62P occurred after reperfusion for 12 h. Immunohistochemical staining showed CD62P significantly deposited on the endothelium after reperfusion. Administration of abciximab (1 mg/kg) effectively improved flap survival rate (P = 0.003), significantly decreased ADP-induced platelet aggregation (P < 0.001), and suppressed CD62P expression on blood platelets (P = 0.002) and its deposition on the flap vessels. CONCLUSION: Abciximab promotion of skin flap survival is due to blocked platelet activation/aggregation and decreased activated-platelet deposition on the vascular endothelium. Thus, administration of a platelet glycoprotein IIb/IIIa receptor antagonist such as abciximab may save the skin flap from reperfusion injury after a long period of ischemia.     

The GP IIb/IIIa inhibitor abciximab (ReoPro) decreases activation and interaction of platelets and leukocytes during in vitro cardiopulmonary bypass simulation.

OBJECTIVE: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response and increases expression of the platelet activation marker P-selectin which mediates binding of platelets to leukocytes. Inhibition of the platelet GP IIb/IIIa receptor during CPB has been shown to protect platelets without increasing bleeding complications and is assumed to reduce the inflammatory response. The aim of this study was to investigate the effect of the GP IIb/IIIa inhibitor abciximab (ReoPro) on the function and interaction of platelets and leukocytes during experimental CPB. METHODS: Heparinized (3 U/ml) fresh whole blood of healthy volunteers was treated before continuous in vitro circulation in a well established CPB model with 3.2 microg/ml abciximab (n=6) or left untreated as control (n=6). Measurements were made before (baseline) and after 30 and 60 min of circulation and comprised: percentage of platelets expressing P-selectin and percentage of platelet-bound leukocytes (flow cytometry), release of the leukocyte activation marker PMN-elastase (ELISA), and platelet and leukocyte counts. RESULTS: Abciximab almost completely prevented a CPB-induced increase of platelet P-selectin and platelet-leukocyte binding after 30 and 60 min of circulation, and significantly inhibited release of PMN-elastase after 30 min of circulation. Furthermore, abciximab significantly inhibited a CPB-induced decrease of platelet and leukocyte counts. CONCLUSIONS: Abciximab inhibits CPB-induced activation, interaction and consumption of platelets and leukocytes in vitro. GP IIb/IIIa inhibition should be considered as a promising approach not only to conserve platelet function but also to inhibit pro-inflammatory events during CPB in vivo.     

Effect of pre-treatment with catecholamines on cold preservation and ischemia/reperfusion-injury in rats

Treatment of organ donors with catecholamines reduces acute rejection episodes and improves long-term graft survival after renal transplantation. The aim of this study was to investigate the effect of catecholamine pre-treatment on ischemia/reperfusion (I/R)- and cold preservation injury in rat kidneys. I/R-injury was induced by clamping the left kidney vessels for 60 min along with a contralateral nephrectomy. Cold preservation injury was induced by storage of the kidneys for 24 h at +4 degrees Celsius in University of Wisconsin solution, followed by syngeneic transplantation. Rats were pre-treated with either dopamine (DA), dobutamine (DB), or norepinephrine (2, 5, and 10 microg/kg/min, each group) intravenously via an osmotic minipump for 24 h before I/R- and cold preservation injury. Pre-treatment with DA (2 or 5 microg/kg/min) and DB (5 microg/kg/min) improved recovery of renal function after I/R-injury and dose dependently reduced mononuclear and major histocompatibility complex class II-positive cells infiltrating the kidney after I/R-injury. One day after I/R-injury, upregulation of transforming growth factor (TGF)-beta 1 and 2 and phosphorylation of p42/p44 mitogen-activated protein kinases was observed in kidneys of animals treated with DA or DB. DA (5 microg/kg/min) and DB (5 microg/kg/min) pre-treatment reduced endothelial cell damage after 24 h of cold preservation. Only DA pre-treatment improved renal function and reduced renal inflammation after 24 h of cold preservation and syngeneic transplantation. Our results demonstrate a protective effect of pre-treatment with catecholamines on renal inflammation and function after I/R- or cold preservation injury. This could help to explain the potent organoprotective effects of catecholamine pre-treatment observed in human kidney transplantation.     

Long-term effects of acute ischemia and reperfusion injury

Ischemia reperfusion (I/R) injury plays a major role in delayed graft function and long-term changes after kidney transplantation. By using different therapeutic strategies to prevent I/R injury in rat models of kidney transplantation we studied relationships between inflammatory cell arrival and adhesion molecule expression. In other rat models for acute renal failure we investigated the effect of up-regulation of protective genes such as heme oxygenase-1 (HO-1) on infiltrating cells, showing that infiltrating cells also contribute to beneficial effects. In order to gain more insight into the complex mechanisms of long-term changes after kidney transplantation, we started a protocol biopsy program to study histologic changes 6, 12, and 26 weeks after transplantation. The following article clarifies some of the complex mechanisms contributing to long-term changes caused by I/R injury.     

Comparison of the effects of desflurane and sevoflurane on the expression of platelet surface glycoproteins in unstimulated and adenosine diphosphate-induced platelets in vitro

STUDY OBJECTIVE: To compare the effects of one minimum alveolar concentration (MAC) desflurane and sevoflurane on the expression of CD42b (glycoprotein [GP] Ib), CD41 (GPIIb), CD61 (GPIIIa), CD62P (P-selectin), and CD63 in both unstimulated and adenosine diphosphate (ADP)-stimulated platelets in vitro. SETTING: University laboratory. SUBJECTS: 15 healthy volunteers. INTERVENTIONS: Platelet-rich plasma was obtained and divided into three groups: platelet-rich plasma exposed to air (group 1); air plus one MAC desflurane (6% vol; group 2), and air plus one MAC sevoflurane (2% vol; group 3), for 40 minutes. Percentage of antigen-positive cells (%(+)) mean channel fluorescence (MCF(Sigma)), and index of platelet activation for positive platelets (IPA(+)) as expression markers for GPIb, GPIIb, GPIIIa, P-selectin, and CD63, were measured. MEASUREMENTS AND MAIN RESULTS: In unstimulated platelets, expression markers for GPIIb and GPIIIa were significantly lower in groups 2 and 3 than group 1 (P < 0.001). P-selectin expression markers were significantly higher in group 2 than in group 1 or group 3 (P < 0.016). CD63 expression markers were significantly lower in group 3 than group 1 (P < 0.016). In ADP-stimulated platelets, expression markers for all glycoproteins were significantly higher in all groups. CONCLUSION: Neither one MAC desflurane nor sevoflurane showed any significant change in ADP-stimulated platelets compared with the control group.     

肝移植术后移植肝组织P-选择素、ICAM-1基因mRNA表达的实验研究

目的：研究移植肝组织P-选择素、ICAM-1基因mRNA表达变化以及供肝交感神经、枯否细胞对mRNA表达的影响。方法：使用氯化钆抑制供肝枯否细胞、六甲胺阻断供肝交感神经，观察肝移植术后4，8，16，24h移植肝P-选择素、ICAM-1 mRNA表达的变化。结果：肝移植术后肝组织P-选择素、ICAM-1基因mRNA表达增高，阻断供肝交感神经和抑制供肝枯否细胞，可下调P-选择素、ICAM-1的mRNA表达。结论：阻断供肝交感神经和抑制供肝枯否细胞能明显下调肝组织P-选择素、ICAM-1基因表达，减轻移植肝的缺血再灌注损伤。     

Inhibition of platelet aggregation by the GPIIb/IIIa antagonist Reopro does not significantly prolong xenograft survival in an ex vivo model

Effects on hyperacute rejection were studied in a discordant model with the platelet GPIIb/IIIa antagonist Reopro. Pig kidneys perfused with human blood survived median 118 min in the Reopro group and 103 min in the controls (P = 0.22). Platelet and leukocyte counts decreased, whereas plasma thrombospondin and soluble as well as platelet membrane P-selectin increased significantly in both groups without significant intergroup differences. beta-Thromboglobulin and myeloperoxidase increased significantly more in the control group than in the Reopro group (P = 0.009 and P = 0.02, respectively). The classical complement pathway was substantially and similarly activated in both groups. Light and electron microscopy revealed arterial thrombi and numerous glomerular platelet aggregates in the control group in contrast to the Reopro group. In conclusion, Reopro reduced platelet aggregation, and platelet and leukocyte activation to some extent, but had no effect on complement activation and did not significantly prolong xenograft survival, even though better preservation of morphology was shown.     

Platelet GPIIb/IIIa is activated and platelet-leukocyte coaggregates formed in vivo during hemodialysis

BACKGROUND/AIM: During hemodialysis, platelets and leukocytes are activated and form platelet-leukocyte coaggregates in which GPIIb/IIIa (CD41/CD61) and CD62P (P-selectin) are involved. However, it is still controversial whether platelet activation and platelet-leukocyte coaggregate formation are dependent on the dialyzer membrane material. METHOD: We examined the appearance of activation-dependent antibody on platelets as an index of platelet activation, and the appearance of platelet-specific antigen on leukocytes as an index of platelet-leukocyte coaggregation, during hemodialysis in 7 patients treated using regenerated cellulose (RC) membrane and next using polysulfone (PS) membrane. In order to reduce the influence of factors other than dialyzer membrane material, this study was conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate. Moreover, flow cytometric techniques with whole blood were employed, which reduce artificial cell activation during the cell or plasma separation procedure. The platelet-specific monoclonal antibodies used in this study were anti-CD61, PAC-1 (which recognizes only the conformationally activated GPIIb/IIIa) and anti-CD62P. RESULTS: Changes in the percentage of PAC-1-positive platelets were significantly greater during hemodialysis with RC than with PS. However, changes in the percentage of CD62P-positive platelets were not significantly different between hemodialysis with RC and PS. Changes in the percentage of CD61- or CD62P-positive leukocytes were significantly greater during hemodialysis with RC than with PS. Although changes in percentage of PAC-1-positive platelets did not parallel those of CD62P-positive platelets during hemodialysis, there was a significant positive correlation between the percentage of CD61-positive leukocytes and the percentage of CD62P-positive leukocytes. CONCLUSION: This study, conducted in a prospective crossover fashion using a pyrogen-free bicarbonate dialysate in order to reduce the influence of factors other than the dialyzer membrane material, demonstrated that both the degrees of GPIIb/IIIa activation and platelet-leukocyte coaggregation were greater during hemodialysis with RC than PS.     

Early expression of adhesion molecules after lung transplantation: evidence for a role of aggregated P-selectin-positive platelets in human primary graft failure.

BACKGROUND: Primary graft failure (PGF) secondary to ischemia-reperfusion injury is the main cause of death in the first month after lung transplantation. The aim of this study was to identify early cellular and immunologic events associated with PGF in human lung transplants. METHODS: Induction of P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) and evaluation of leukocytes and platelets accumulation were investigated in 18 post-reperfusion surgical specimens of lung allografts by an immunohistochemical technique. RESULTS: Selectins were restricted to the venular plexus after reperfusion as in the normal lung, whereas ICAM-1 was induced in all cases on alveolar capillaries. Numerous polymorphonuclear cells (18 of 18 cases) and aggregated platelets (7 of 18 cases) were identified along the venular plexus after reperfusion. Compared with the other patients, those with aggregated P-selectin-positive platelets were characterized by a longer duration of mechanical ventilation (p < 0.01), a lower PaO2/FiO2 ratio (p < 0.01) and the presence of radiologic edema (p < 0.05) within the first 3 post-operative days. CONCLUSIONS: We showed in the reperfused lung a distinct expression of adhesion molecules on venous and capillary pulmonary endothelia that may influence the role of leukocytes and platelets during the early course of transplantation. Furthermore, the knowledge of an association between the presence of P-selectin-positive platelet aggregates and PGF criteria might have implications for graft management and therapeutic strategies.     

Apoptosis and regeneration of sinusoidal endothelial cells after extended cold preservation and transplantation of rat liver

BACKGROUND: Sinusoidal endothelial cells (SECs) are particularly susceptible to cold ischemia-reperfusion (I/R) injury. We have examined the process of injury and recovery of graft after cold-preserved liver transplantation, with special focus on the proliferation of SECs and regulatory mechanisms involved. METHODS: Male SD rats were divided into two groups according to length of cold preservation time in University of Wisconsin [UW] solution of graft: UW1h group and UW12h group. Graft function, incidence of apoptosis, proliferation of SECs and the expression of related regulatory factors were assessed after orthotopic liver transplantation (OLT). RESULTS: SECs are more sensitive to apoptosis induced by cold I/R injury compared with hepatocytes. Using bromodeoxyuridine and rat endothelial cell antigen-1 double immunostaining assay, SECs exhibited a delayed proliferation in comparison with hepatocytes, reaching a peak at 72 hr in UW1h group and 96 hr in UW12h group, respectively. Vascular endothelial growth factor increased at 24 hr after reperfusion, and peaked at 72 hr in both groups. Flt-1 and flk-1 expression was found to be mainly limited to SECs, with a peak in expression occurring between 72 and 96 hr, which coincided with the peak in SEC proliferation in UW1h group. However, flt-1 was found to be reduced significantly at any time throughout the experiments in UW12h group compared to sham. CONCLUSION: The delayed recovery of rat liver after extended cold preservation and transplantation correlates with a retarded regeneration of SECs due to increased apoptosis and reduced expression of flt-1. These results suggest that SECs play an important role in cold-preserved liver transplantation.     

Xenon does not affect human platelet function in vitro

We sought to determine whether xenon affects platelet glycoprotein expression and platelet-related hemostasis in vitro at a clinically relevant concentration. Human whole blood was stimulated with either adenosine diphosphate or the thrombin receptor agonist peptide (TRAP)-6 after incubation with 65% xenon. Halothane at 2 minimum alveolar anesthetic concentration was used as a positive control. Platelet function and activation were evaluated with two-color flow cytometry. The expression of the platelet glycoproteins GPIIb/IIIa, GPIb, and P selectin were detected with fluorochrome-conjugated monoclonal antibodies. In vitro measurement of platelet-related hemostasis under conditions of high shear stress was performed in citrated whole blood with a platelet function analyzer (PFA-100((R))) by using collagen/epinephrine and collagen/adenosine diphosphate cartridges. Xenon did not affect basal or agonist-induced expression of platelet membrane glycoproteins, activation-dependent conformational changes of the GPIIb/IIIa receptor, expression of P selectin, or PFA closure times. In contrast, halothane reduced TRAP-6-induced activation of the GPIIb/IIIa complex. Furthermore, collagen/epinephrine-induced PFA closure time was significantly prolonged. These results demonstrate that xenon does not affect the unstimulated or agonist-induced platelet glycoprotein expression, activation of GPIIb/IIIa, or platelet-related hemostasis.     

Phosphodiesterase III inhibition affects platelet-monocyte aggregate formation depending on the axis of stimulation

OBJECTIVE: The purpose of this study was to investigate the effect of the phosphodiesterase (PDE) type 3 inhibitor milrinone on the adhesion of platelets to monocytes in vitro. DESIGN: Prospective study. SETTING: University experimental laboratory. PARTICIPANTS: Ten healthy volunteers. INTERVENTIONS: Whole blood was incubated with 1, 10, or 100 micromol/L of milrinone. After stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or adenosine-5-diphosphate (ADP), platelet-monocyte adhesion and CD11b, PSGL-1, GPIIb/IIIa, and P-selectin expression were measured by flow cytometry. MEASUREMENTS AND RESULTS: The formation of platelet-monocyte conjugates after PDE3 inhibition depended on the type of stimulation. In unstimulated and FMLP-stimulated blood platelet monocytes, aggregation was enhanced by increasing concentrations of milrinone. This augmentation was accompanied by a rise in P-selectin expression in platelets. In ADP-stimulated blood the number of platelet-monocyte aggregates decreased with increasing concentrations of milrinone. Concurrent with the reported antiinflammatory properties of PDE-inhibition, an inhibition of CD11b expression was found in monocytes after stimulation with FMLP. In contrast, in unstimulated samples lower concentrations of milrinone caused an increase in CD11b. CONCLUSIONS: These findings suggest that the effects of PDE3 inhibition on platelets and monocytes are modified by the type of stimulation and only partially suppress the inflammatory response of platelets and monocytes. The increase in platelet-monocyte conjugates in unstimulated and FMLP-stimulated blood suggested that PDE3 inhibition may also trigger proinflammatory reactions.     

Detailed Analysis of Mucosal Restoration of the Small Intestine After the Cavitary Two-Layer Cold Storage Method

Small bowel transplantation (SBT) is associated with a high incidence of infectious complications because of ischemia/reperfusion (I/R) mucosal injury concomitant with potent immunosuppression. In this study, we evaluated whether the cavitary two-layer method (cTLM) could reduce I/R injury and allow early mucosal restoration, particularly after prolonged preservation and transplantation. Canine heterotopic segmental SBT was performed immediately without preservation (group 1), after 24-h preservation in UW solution (group 2) or by the cTLM (group 3). The graft samples were taken 1 h after reperfusion and on days 1, 4 and 7. We assessed graft mucosa with detailed microscopic and electromicroscopic analyses. In Group 3, histological injury and cell apoptosis after transplantation were significantly alleviated and rapidly recovered to a similar level of group 1. The mucosal restoration was morphologically completed within 4 days. In contrast, in group 2, more pronounced mucosal injury and delayed recovery were noted. Crypt cell proliferation activity was well maintained in groups 1 and 3 throughout the experimental period. Our ultrastructural analysis suggested that mitochondrial integrity achieved by the cTLM was a basal mechanism under the prompt mucosal restoration. The cTLM could reduce I/R injury, facilitate mucosal regeneration and restore the nearly normal structure early after SBT.