P-selectin on activated platelets and stimulated endothelial cells mediat cell adhesion with monocytes and neutrophils. Since activated platelets induce tissue factor on mononuclear leukocytes, we examined the effect of P-selectin on the expression of tissue factor activity in monocytes.Purified P-selectin stimulated tissue factor expression on mononuclear leukocytes in a dose-dependent manner. Chinese hamster ovary (CHO) cells expressing P-selectin stimulated tissue factor procoagulant activity in purified monocytes, whereas untransfected CHO cells and CHO cells expressing E-selectin did not. Anti-P-selectin antibodies inhibited the effects of purified P-selectin and CHO cells expressing P-selectin on monocytes. Incubation of CHO cells expressing P-selectin with monocytes leads to the development of tissue factor mRNA in monocytes and to the expression of tissue factor antigen on the monocyte surface. These results indicate that P-selectin upregulates the expression of tissue factor on monocytes as well as mediates the binding of platelets and endothelial cells with monocytes and neutrophils. The binding of P-selectin to monocytes in the area of vascular injury may be a component of a mechanism that initiates thrombosis. stimulation of these cells by agonists such as thrombin, P-selectin is phosphorylated (26,27) and rapidly translocated to the plasma membrane (23). P-selectin is a lectin that binds to lineage-specific carbohydrates on the surface of monocytes and neutrophils (28)(29)(30). This protein binds to a mucinlike glycoprotein PSGL-1 that must be properly glycosylated to retain functional properties as the P-selectin ligand (31). P-selectin on platelets mediates the accumulation of leukocytes into the growing thrombus during experimental thrombosis in vivo (32). Inhibitory antibodies that block the interaction of P-selectin on platelets with the P-selectin ligand on leukocytes inhibit the uptake of leukocytes into the thrombus and inhibit the magnitude of thrombus formation. These experiments have demonstrated that P-selectin mediates monocyte and neutrophil interaction with activated platelets in vitro and in vivo. The potential exists for stimulation of leukocyte effector function by P-selectin binding. To evaluate this potential, we have examined the ability of P-selectin to upregulate tissue factor expression on monocytes. In the current study, we demonstrate that P-selectin induces the expression of tissue factor on monocytes exposed to P-selectin. Blood clotting is a host defense mechanism that, in parallel with the inflammatory and repair responses, preserves the integrity of the vascular system after tissue injury (1). Platelets, leukocytes, and endothelial cells are among the cellular components critical for this process. The plasma blood clotting proteins participate in a molecular cascade in which tissue injury activates blood coagulation, leading to the formation of a fibrin clot (2). The response to vascular injury culminates in the formation of a platelet plug, the deposition of ...

Abstract-Plasma plasminogen activator inhibitor type 1 (PAI-1) increases in diabetes, and this might contribute to decreased fibrinolysis and accelerated atherosclerosis. Increased PAI-1 levels in the vessel wall could decrease local fibrinolysis and elevate thrombus formation and the unfavorable evolution of atherosclerotic plaques. High glucose increases PAI-1 synthesis in arterial wall cells in culture, and aortic wall PAI-1 levels have been found to be elevated in diabetic animals. However, arterial wall PAI-1 levels have not been investigated in diabetic subjects. Therefore, the aim of this study was to determine the effect of diabetes on PAI-1 levels in the arterial wall. Blood samples and small tissue specimens from the mammary artery were obtained from 11 diabetic and 10 nondiabetic subjects who underwent coronary artery bypass graft surgery. PAI-1 antigen localization in the arterial wall was obtained by immunohistochemistry and was read by laser scanning confocal microscopy; plasma fibrinolytic activity was measured by lysis of fibrin plates; and PAI-1 activity was assessed by a chromogenic method. PAI-1-related immunofluorescence was increased in the arterial wall of diabetic patients, whereas plasma fibrinolysis was reduced. These data provide evidence that diabetes is associated with increased PAI-1 in the arterial wall. This might be an important factor for increased cardiovascular risk and unfavorable plaque evolution in diabetes. Key Words: diabetes mellitus Ⅲ coronary disease Ⅲ fibrinolysis Ⅲ arteries Ⅲ plasminogen activator inhibitor type 1 T ype II diabetes mellitus is associated with increased morbidity and mortality due to atherosclerosis. 1,2 Although several of the traditional risk factors for cardiovascular disease (CVD) are increased in type II diabetes, these factors account for no more than half of the observed increased risk for CVD. 3 Because of the importance of acute thrombosis in the pathogenesis of CVD, decreased fibrinolytic capacity has been proposed as an additional risk factor for CVD, and indeed, levels of plasminogen activator inhibitor type 1 (PAI-1), a biochemical marker of impaired fibrinolysis, have been shown to be correlated with reinfarction rates in subjects with previous myocardial infarction. 4 Furthermore, plasma PAI-1 levels have been shown to be correlated with hyperinsulinemia and/or insulin resistance, 5,6 so that increased plasma PAI-1 levels are now considered as one of the features of the insulin resistance syndrome. 7 Increased plasma PAI-1 activity has also been reported, although inconsistently, 8 in subjects with type II diabetes; this has mostly been attributed to the insulin resistance/hyperinsulinemia that often occurs in these patients. 9 -11 On the other hand, several lines of evidence suggest that hyperglycemia, per se, could also induce impaired fibrinolysis. Thus, there is evidence that high glucose decreases fibrinolytic capacity in cultured cells, 12,13 and we have recently shown, in a rat model, that increased PAI-1 levels are associated wit...

Decreased plasma fibrinolysis may contribute to accelerated atherothrombosis in diabetes. To observe whether hyperglycemia and hyperinsulinemia, common findings in type 2 diabetes, acutely affect plasma fibrinolysis in vivo, we evaluated plasma fibrinolysis (lysis of fibrin plates, free PAI-1 activity and t-PA activity) in the rat after a hyperglycemic euinsulinemic clamp (n=8), an euglycemic hyperinsulinemic clamp (n=7) or a saline infusion (n=15). Plasma fibrinolytic activity was sharply reduced after both the hyperglycemic and hyperinsulinemic clamps as compared to the respective controls (mean lysis areas on the fibrin plate, 139+/-21 vs. 323+/-30 mm2, p<0.001; 78+/-27 vs. 312+/-27 mm2 p<0.001, respectively). Plasma PAI-1 activity was greater after both hyperglycemic and hyperinsulinemic clamps as compared to saline infusion (6.6+/-2.6 vs. 1.6+/-0.6 IU/ml, p<0.001; 26+/-4 vs. 1.3+/-0.7 IU/ml, p<0.0001, respectively). Plasma t-PA activity was significantly reduced both after the hyperglycemic (0.36+/-0.15 vs. 2.17+/-0.18 IU/ml in controls, p<0.001) and the hyperinsulinemic (0.3+/-0.1 vs. 2.3+/-0.3 IU/ml in control, p<0.001) clamps. These data show that in vivo both acute hyperglycemia and acute hyperinsulinemia can decrease plasma fibrinolytic potential and that this is due to increased plasma PAI-1 and decreased free t-PA activities.

Background-Impaired insulin-mediated vasodilation might contribute to vascular damage in insulin-resistant states. Little is known about insulin regulation of nitric oxide (NO) synthesis in insulin-resistant cells. The aim of this work was to investigate insulin regulation of NO synthesis in human umbilical vein endothelial cells (HUVECs) carrying the IRS-1 gene G972R variant, known to be associated with impaired insulin activation of the PI3-kinase (PI3-K) pathway in transfected cells. Methods and Results-HUVECs were screened for the presence of the G972R-IRS-1 (HUVEC-G972R) variant by restriction fragment length polymorphisms. After 24-hour exposure to 10 Ϫ7 mol/L insulin, endothelial NO synthase (eNOS) mRNA (reverse transcription-polymerase chain reaction), eNOS protein levels (Western blotting), and NOS activity (conversion of [ 3 H]arginine into [ 3 H]citrulline) were increased in wild-type HUVECs (HUVEC-WT), whereas they did not change from baseline in HUVEC-G972R. Compared with HUVEC-WT, in HUVEC-G972R after 2 and 10 minutes of insulin stimulation, IRS-1-associated PI3-K activity was reduced by 47% and 32%, respectively; Akt phosphorylation was decreased by 40% at both time points; and eNOS-Ser1177 phosphorylation was reduced by 38% and 51%, respectively. In HUVEC-WT, eNOS-Thr495 phosphorylation decreased after insulin stimulation. In contrast, in HUVEC-G972R, eNOS-Thr495 phosphorylation increased after insulin stimulation and was 40% greater than in HUVEC-WT. Conclusions-Our

Proliferative modification of vascular smooth muscle cell (vSMC) and impaired bioavailability of nitric oxide (NO) have both been proposed among the mechanisms linking diabetes and atherosclerosis. However, diabetes induced modifications in phenotype and nitric oxide synthase(s) (NOS) expression and activity in vSMC have not been fully characterized. In this study, cell morphology, proliferative response to serum, alpha-SMactin levels, eNOS expression and activity, cGMP intracellular content, and superoxide anion release were measured in cultures of vSMC obtained from aorta medial layer of ten diabetic (90% pancreatectomy, DR) and ten control (sham surgery, CR) rats. Vascular SMC from DR showed a less evident "hill and valley" culture morphology, increased growth response to serum, greater saturation density, and lower levels of alpha-SMactin. In the same cells, as compared to CR cells, eNOS mRNA levels and NOS activity were increased, while intracellular cGMP level was lower and superoxide anion production was significantly greater. These data indicate that chronic hyperglycemia might induce, in the vascular wall, an increased number of vSMC proliferative clones which persist in culture and are associated with increased eNOS expression and activity. However, upregulation of eNOS and increased NO synthesis occur in the presence of a marked concomitant increase of O(2-) production. Since NO bioavailability, as reflected by cGMP levels, was not increased in DR cells, it is tempting to hypothesize that the proliferative phenotype observed in DR cells is associated with a redox imbalance responsible quenching and/or trapping of NO, with the consequent loss of its biological activity.

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12-Hydroxyeicosatetraenoic acid (12-HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P-selectin-stimulated monocytes in a time-and dose-dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P-selectin cDNA. Both 5-HETE and leukotriene C 4 are inactive in this system. Furthermore, the effect is not dependent on non-specific monocyte adhesion, since monocytes incubated with CHO cells expressing E-selectin do not express tissue factor, either in the presence or in the absence of 12-HETE. These results show that 12-HETE is a cofactor for the expression of tissue factor by monocytes.z 1998 Federation of European Biochemical Societies.