In vivo and protease-activated receptor-1-mediated platelet activation but not response to antiplatelet therapy predict two-year outcomes after peripheral angioplasty with stent implantation.

Data linking the response to antiplatelet therapy with clinical outcomes after angioplasty and stenting for lower extremity artery disease (LEAD) are scarce. Moreover, associations of in vivo and thrombin-inducible platelet activation with the occurrence of adverse events have not been investigated in these patients, so far. We therefore assessed clinical outcomes and on-treatment platelet reactivity by four test systems in 108 patients receiving dual antiplatelet therapy after infrainguinal angioplasty and stenting for LEAD. Further, in vivo and thrombin receptor-activating peptide (TRAP)-6-inducible glycoprotein (GP) IIb/IIIa activation and P-selectin expression were measured as sensitive parameters of platelet activation. The primary endpoint was defined as the composite of atherothrombotic events and target vessel restenosis or reocclusion. Residual platelet reactivity to adenosine diphosphate and arachidonic acid was similar between patients without and with adverse outcomes within two-year follow-up (all p>0.05). Further, the occurrence of clinical endpoints did not differ significantly between patients without and with high on-treatment residual platelet reactivity by all test systems (all p>0.05). In contrast, in vivo and TRAP-6-inducible platelet activation were significantly more pronounced in patients with subsequent adverse events (all p<0.05), and high levels of platelet activation were independent predictors of the primary endpoint (adjusted hazard ratios: 3.5 for high in vivo activated GPIIb/IIIa, 2.9 for high TRAP-6-inducible activated GPIIb/IIIa, 2.3 for high in vivo P-selectin, and 3 for high TRAP-6-inducible P-selectin; all p<0.05). In conclusion, in vivo and protease-activated receptor-1-mediated platelet activation predict two-year clinical outcomes in stable patients undergoing angioplasty and stenting for LEAD.

Adenosine diphosphate-inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel.

BACKGROUND: Until recently, there were hardly any data on the antiplatelet effect of clopidogrel in advanced age. Like other metabolic processes, the conversion of clopidogrel to its active metabolite may be impaired in older patients, leading to high on-treatment residual ADP-inducible platelet reactivity. OBJECTIVE: To investigate the age dependency of clopidogrel-mediated platelet inhibition. PATIENTS AND METHODS: This was a prospective observational study. We determined adenosine 5'-diphosphate (ADP)-inducible platelet reactivity using light transmission aggregometry (LTA) and the VerifyNow P2Y12 assay in 191 patients on dual antiplatelet therapy after angioplasty and stenting for cardiovascular disease. RESULTS: ADP-inducible platelet reactivity increased linearly with age after adjustment for cardiovascular risk factors, type of intervention, medication, C-reactive protein (CRP) and renal function [using LTA 0.36% of maximal aggregation per year, 95% CI 0.08-0.64%, P = 0.013; using the VerifyNow P2Y12 assay 3.2 P2Y12 reaction units (PRU) per year, 95% CI 1.98-4.41 PRU, P < 0.001]. ADP-inducible platelet reactivity was significantly higher in patients aged 75 years or older compared with younger patients (P = 0.003 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). Further, high on-treatment residual ADP-inducible platelet reactivity was significantly more common among patients aged 75 years or older (P = 0.02 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). CONCLUSION: ADP-inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel. The clinical implications of these findings need to be addressed in future trials.

Inflammatory cytokines interleukin-6 and oncostatin m induce plasminogen activator inhibitor-1 in human adipose tissue.

BACKGROUND: Adipose tissue is a prominent source of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of plasminogen activation. Increased PAI-1 expression acts as a cardiovascular risk factor, and plasma levels of PAI-1 strongly correlate with body mass index (BMI). Elevated serum levels of interleukin-6 (IL-6), an inflammatory cytokine and a member of the glycoprotein 130 (gp130) ligand family, are found in obese patients and might indicate low-grade systemic inflammation. Another gp130 ligand, oncostatin M (OSM), upregulates PAI-1 in cardiac myocytes, astrocytes, and endothelial cells. We used tissue explants and primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether IL-6 and OSM affect PAI-1 expression in fat. METHODS AND RESULTS: Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in PAI-1 production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte differentiation was induced by hormone supplementation. All cell types expressed receptors for IL-6 and OSM and produced up to 12-fold increased levels of PAI-1 protein and up to 9-fold increased levels of PAI-1 mRNA on stimulation with IL-6 and OSM. AG-490, a janus kinase/signal transducer and activator of transcription inhibitor, abolished the OSM-dependent PAI-1 induction almost completely. CONCLUSIONS: We have for the first time established a link between the gp130 ligands, the proinflammatory mediators IL-6 and OSM, and the expression of PAI-1 in human adipose tissue. Thus, we speculate that IL-6 and OSM, by upregulating PAI-1 in adipose tissue, can contribute to the increased cardiovascular risk of obese patients.

Impact of weight loss on inflammatory proteins and their association with the insulin resistance syndrome in morbidly obese patients.

OBJECTIVE: Obesity is closely linked to the insulin resistance syndrome (IRS), type 2 diabetes, and cardiovascular disease, the primary cause of morbidity and mortality in these patients. Elevated levels of C-reactive protein (CRP) and interleukin-6 (IL-6), indicating chronic subclinical inflammation, have been associated with features of the IRS and incident cardiovascular disease. METHODS AND RESULTS: We studied the cross-sectional and longitudinal relation of CRP, IL-6, and tumor necrosis factor-alpha (TNF-alpha) with features of the IRS in 37 morbidly obese patients with different stages of glucose tolerance before and 14 months after gastric surgery. Weight loss after gastric surgery induced a significant shift from diabetes (37% vs 3%) to impaired glucose tolerance (40% vs 33%) and normal glucose tolerance (23% vs 64%). The baseline concentration of IL-6 was correlated with TNF-alpha (r=0.59, P<0.01) and CRP (r=0.44, P<0.05) levels. TNF-alpha, IL-6, and CRP were significantly correlated with insulin resistance estimated by the homeostatic model assessment (r=0.48, P<0.05; r=0.56, P<0.01; and r=0.35, P<0.05, respectively). Concentrations of CRP and IL-6 decreased after weight loss (median, 8.6 and interquartile range, 2.7/14.5 vs 2.5 and 1.2/4.1 mg/L; P<0.006, and 5.13 and 2.72/12.15 vs 3.95 and 1.97/5.64 pg/mL, P<0.02, respectively), whereas serum levels of TNF-alpha remained unchanged (8.6 and 6.3/18.8 vs 11.7 and 5.8/17.2 pg/mL; NS.). Multiple regression analysis revealed that the decrease in insulin resistance remained independently and significantly correlated with the decrease in IL-6 concentrations (P<0.01) and the decrease in body mass index with the decrease in CRP (P<0.05), respectively. CONCLUSIONS: Weight loss in morbidly obese patients induces a significant decrease of CRP and IL-6 concentrations in association with an improvement of the IRS.

Effect of intradermal tumor necrosis factor-alpha-induced inflammation on coagulation factors in dermal vessel endothelium. An in vivo study of human skin biopsies.

Inflammatory mediators were shown to exert procoagulant effects on cultured human endothelial cells (EC). In the present study the effect of intradermal application of tumor necrosis factor-alpha (TNF-alpha) on the expression of factors involved in regulation of coagulation at the EC surface, i.e. tissue factor (TF), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was studied in humans in vivo. The endothelial expression of these factors was evaluated immunohistochemically in biopsies taken after intradermal application of 5000 U TNF-alpha in 8 healthy volunteers. After 6 and 22 h biopsies were taken from the injection sites. At TNF-alpha injected sites typical inflammatory changes. e.g. EC upregulation of adhesion molecules and accumulation of leukocytes were detected. In parallel we could document EC expression of TF, downregulation of TM and depletion of tissue factor pathway inhibitor (TFPI) in inflamed areas. Early depletion of endothelial IkappaB alpha at the site of inflammation after application of TNF-alpha points to an activation of the NF-kappaB pathway. Our data suggest that, as shown in in vitro experiments, TNF-alpha activates the NF-kappaB pathway and induces specific procoagulant changes of EC due to expression of TF, down-regulation of TM and depletion of TFPI in vivo in humans. This procoagulant shift in the haemostatic balance on the cell surface, caused by TNF-alpha-induced inflammation, is likely to contribute to thrombosis associated with tissue inflammation in humans.

Expression of human thrombomodulin cofactor activity in porcine endothelial cells.

BACKGROUND: Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. METHODS AND RESULTS: Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses. CONCLUSIONS: Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Combined heart and kidney transplantation using a single donor: a single center's experience with nine cases.

BACKGROUND: Simultaneous double-organ transplants comprising various organ combinations have become frequent. The purpose of this article is to report on a single center's experience of simultaneous heart and kidney transplantation (HNTX) with particular emphasis on selection criteria and patient outcome. METHODS: From September 1990 to January 1997, nine patients underwent HNTX, receiving both grafts from a single donor selected on ABO blood group compatibility and a negative lymphocytotoxic crossmatch, but without regard to HLA-antigen matching. RESULTS: One patient died of acute humoral rejection of the cardiac graft shortly after surgery. Eight patients are alive and well and have normal cardiac and renal function at a mean follow-up of 44+/-28 months. CONCLUSION: HNTX offers a compelling therapeutic solution in the treatment of advanced cardiac and renal failure in carefully selected patients. Because the heart and kidney rejection episodes were independent of each other, rejection surveillance should be carried out separately for each transplanted organ.

Xenogeneic endothelial cells activate human prothrombin.

BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Expression of the monoclonal antibody HECA-452 defined E-selectin ligands in Langerhans cell histiocytosis.

The cutaneous lymphocyte associated antigen (CLA) recognized by the monoclonal antibody (moAb) HECA-452 plays a major role in the homing of lymphocyte subpopulations to the skin by binding to E-selectin on dermal microvessels. The factors responsible for the immigration of Langerhans cells (LC) and their precursors into the skin are still unknown, but because normal resting LC are also capable of expressing CLA, the antigen was proposed as a candidate LC-homing structure. To gain insight into these mechanisms, the expression of HECA-452 on neoplastic LC within and outside the skin was investigated in paraffin-embedded sections from 44 patients with localized and disseminated forms of Langerhans cell histiocytosis (LCH) presenting with proliferating cells positive for CD45, CD1a, S100 and HLA-DR. Irrespective of the clinical presentation or the type of organ involved, HECA-452 positive LC were detected in all biopsies tested (range 5->90%). The most prominent HECA-452 reactivity was observed in skin lesions and in areas with accumulations of eosinophilic granulocytes. Our data provide evidence for heterogeneous expression of sLex/sLea structures in various stages of activated and/or differentiated LCH cells. Remarkably, CLA-antigen expression on LCH-cells was not restricted to cutaneous sites. In view of recent findings on the expression of HECA-452 on resting epidermal LC, our data are compatible with the view that local cytokine production by keratinocytes or cells from the surrounding infiltrate induce and/or modulate CLA expression on LC in both cutaneous and extra-cutaneous sites.