Distinct antithrombotic consequences of platelet glycoprotein Ibalpha and VI deficiency in a mouse model of arterial thrombosis.

BACKGROUND: Collagen and von Willebrand factor (VWF) are considered essential to initiate platelet deposition at sites of vascular injury, but their respective roles remain to be elucidated. METHODS: We used a model of carotid artery thrombosis induced by a ferric chloride injury to compare the time to first occlusion and occlusion rate at 25 min postinjury in mice lacking the collagen receptor, glycoprotein (GP) VI, or the ligand-binding domain of the VWF receptor, GP Ibalpha. RESULTS: In normal mice used as controls (n = 12), a complete obstruction of blood flow developed within 8.05 +/- 0.47 min (mean +/- SEM), and the occlusion rate was 100%. The results were variable in 26 GP VI(-/-) mice. The artery never occluded in eight mice, but the time to first occlusion in the remaining 18 (8.36 +/- 0.27 min) was not different from normal (P = 0.556). Nonetheless, the occlusion rate was 42%, because in seven mice the occluded artery reopened and stayed patent at 25 min. In contrast, the artery never occluded in 12 mice lacking GP Ibalpha. In ex vivo perfusion experiments, GP VI(-/-) platelets failed to form thrombi onto collagen type I fibrils, but formed thrombi of normal size when exposed to endothelial or fibroblast extracellular matrix. CONCLUSIONS: Absence of GP Ibalpha function has a more profound antithrombotic effect in vivo than absence of the GP VI-dependent pathway of collagen-induced adhesion/activation. Components of the extracellular matrix may elicit a thrombogenic response in the absence of GP VI but not GP Ibalpha.

Thrombotic phenotype of mice with a combined deficiency in plasminogen activator inhibitor 1 and vitronectin.

OBJECTIVE: The role of vitronectin (VN) in thrombosis is not fully understood, primarily because this adhesive glycoprotein not only stabilizes plasminogen activator inhibitor 1 (PAI-1) and thus protects fibrin from premature lysis, but also because it binds to platelet integrins and may influence platelet aggregation. The absence of quantitative approaches to characterize the thrombi formed in animal models under different conditions further complicates this analysis. METHODS: In this report, we describe a more comprehensive approach to assess the stability of thrombi formed in mice deficient in PAI-1 (PAI-1(-/-)), VN (VN(-/-)) or both (PAI-1(-/-)/VN(-/-)). RESULTS: We observed that all deficient mice developed unstable thrombi compared with wild type (WT) mice. Thus, only 31% of the thrombi formed in WT mice were unstable compared with 74% of PAI-1(-/-), 80% of VN(-/-), and 87% of PAI-1(-/-)/VN(-/-) mice. In this regard, the average number of emboli per WT mouse was significantly lower (0.55) compared with VN(-/-) (2.66), PAI-1(-/-) (2.1), and VN(-/-)/PAI-1(-/-) (2.35) mice. Finally, the total duration of complete vascular occlusion was higher and the rate of vascular patency was lower in the WT mice compared with the deficient mice. CONCLUSIONS: Taken together, these observations indicate that the thrombotic phenotype of mice with a combined deficiency in PAI-1 and VN does not differ significantly from the phenotype of mice with deficiencies in only PAI-1 or VN. This observation suggests that PAI-1 and VN may influence thrombus stability by regulating a common pathway.

Obesity enhances the induction of plasminogen activator inhibitor-1 by restraint stress: a possible mechanism of stress-induced renal fibrin deposition in obese mice.

BACKGROUND AND OBJECTIVES: Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanism is still unknown. We have begun to investigate the expression of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue thrombosis, using restraint-stressed obese mice. METHODS AND RESULTS: We analyzed the expression of PAI-1 after restraint (immobilization) stress in genetically obese mice in comparison with their lean counterparts. Dramatic increases in PAI-1 antigen in plasma and in tissue extracts were observed in the obese mice exposed to restraint stress. The induction of PAI-1 mRNA by stress in the tissues was also pronounced in the stressed obese mice as compared with the lean mice, especially in the hearts and adipose tissues. In situ hybridization analysis revealed that strong signals for PAI-1 mRNA were localized in the adipocytes, cardiovascular endothelial cells, and renal glomerular cells of the stressed obese mice. Histological examination revealed that renal glomerular fibrin deposition was detected only in the obese mice after 2 h of restraint stress. CONCLUSIONS: Obesity enhances the stress-mediated PAI-1 induction in the blood and tissues. This phenomenon may be associated with the increased risk of stress-induced renal fibrin deposition in obese subjects.

The leptin receptor system of human platelets.

Obesity is associated with elevated levels of leptin in the blood. Elevated leptin is a risk factor for thrombosis in humans, and leptin administration promotes platelet activation and thrombosis in the mouse. The current study examines the effect of leptin on human platelets, and provides initial insights into the nature of the leptin receptor on these platelets. Leptin potentiated the aggregation of human platelets induced by low concentrations of ADP, collagen and epinephrine. However, the response varied significantly between donors, with platelets from some donors (approximately 40%) consistently responding to leptin (responders) and those from other donors (approximately 60%) never responding (non-responders). Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that platelets from both groups only express the signaling form of the leptin receptor, and that responder platelets express higher levels of this receptor than non-responders. Ligand-binding assays demonstrate specific, saturable binding of leptin to platelets from both groups with apparent K(d) values of 76 +/- 20 nM for responders and 158 +/- 46 nM for non-responders. Thus, the decreased sensitivity of non-responder platelets to leptin does not result from the absence of the signaling form of this receptor, but may reflect differences in its level of expression and/or affinity for leptin. These preliminary studies demonstrate that platelets are a major source of leptin receptor in the circulation, and suggest that leptin-responsive individuals may have a higher risk for obesity-associated thrombosis than non-responsive individuals.

Regulation of tissue factor gene expression in obesity.

Altered expression of proteins of the fibrinolytic and coagulation cascades in obesity may contribute to the cardiovascular risk associated with this condition. In spite of this, the zymogenic nature of some of the molecules and the presence of variable amounts of activators, inhibitors, and cofactors that alter their activity have made it difficult to accurately monitor changes in the activities of these proteins in tissues where they are synthesized. Thus, as a first approach to determine whether tissue factor (TF) expression is altered in obesity, this study examined changes in TF mRNA in various tissues from lean and obese (ob/ob and db/db) mice. TF gene expression was elevated in the brain, lung, kidney, heart, liver, and adipose tissues of both ob/ob and db/db mice compared with their lean counterparts. In situ hybridization analysis indicated that TF mRNA was elevated in bronchial epithelial cells in the lung, in myocytes in the heart, and in adventitial cells lining the arteries including the aortic wall. Obesity is associated with insulin resistance and hyperinsulinemia, and administration of insulin to lean mice induced TF mRNA in the kidney, brain, lung, and adipose tissue. These observations suggest that the hyperinsulinemia associated with insulin-resistant states, such as obesity and noninsulin-dependent diabetes mellitus, may induce local TF gene expression in multiple tissues. The elevated TF may contribute to the increased risk of atherothrombotic disease that accompanies these conditions.

Leptin-dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity.

Obesity is associated with increased cardiovascular morbidity and mortality and with elevated circulating levels of the satiety factor leptin. This study provides evidence for a direct link between leptin and the risk for thrombotic complications in obese individuals. For example, although arterial injury provokes thrombosis in both lean and obese (ob/ob) mice, the time to complete thrombotic occlusion is significantly delayed in the ob/ob mice, and the thrombi formed are unstable and frequently embolize. The ob/ob mice lack leptin, and intraperitoneal administration of leptin to these mice before injury restores the phenotype of lean mice by shortening the time to occlusion, stabilizing the thrombi, and decreasing the patency rate. The thrombi that form when leptin receptor-deficient obese (db/db) mice are injured also are unstable. However, in this instance, leptin has no effect. Platelets express the leptin receptor, and leptin potentiates the aggregation of platelets from ob/ob but not db/db mice in response to known agonists. These results reveal a novel receptor-dependent effect of leptin on platelet function and hemostasis and provide new insights into the molecular basis of cardiovascular complications in obese individuals. The results suggest that these prothrombotic properties should be considered when developing therapeutic strategies based on leptin.

Tissue distribution of factor VIII gene expression in vivo--a closer look.

Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin.

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.

A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin.

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.

Plasminogen activator inhibitor-1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice.

BACKGROUND: The origin and contribution of plasminogen activator inhibitor-1 (PAI-1) and its cofactor vitronectin (VN) to arterial thrombosis/thrombolysis in vivo is controversial. METHODS AND RESULTS: Ferric chloride was used to induce carotid artery injury in 97 wild-type (WT), 84 PAI-1-/-, and 84 VN-/- mice. Complete thrombotic occlusion was observed in 70% of PAI-1-/- mice versus 92% of WT (P:<0.001) and 87% of VN-/- (P:=0.015) mice. In vessels that occluded, mean times to occlusion were significantly longer in PAI-1-/- than in WT or VN-/- mice. The initial thrombotic response of VN-/- mice was similar to that of WT mice, but their thrombi were unstable and frequently embolized. As a result, the patency rate of carotid vessels 30 minutes after injury was as high in VN-/- mice (36%) as in PAI-1-/- mice (which demonstrate progressive thrombolysis) and significantly higher than that of WT mice (12%; P:=0.013). Histochemical and reverse transcription-polymerase chain reaction studies revealed an early upregulation of PAI-1 mRNA and protein expression in the thrombus and the vessel wall, which persisted for >/=1 week. VN protein also accumulated after injury, but VN mRNA levels remained low at all times. CONCLUSIONS: PAI-1 and VN participate in the thrombotic response to arterial injury by preventing premature thrombus dissolution and embolization. The accumulation of PAI-1 in the thrombus/vessel wall after injury may result, at least in part, from local synthesis, whereas the VN protein appears to be derived from plasma.

The prothrombotic effects of leptin possible implications for the risk of cardiovascular disease in obesity.

Human obesity is associated with leptin resistance, elevated leptin levels in the circulation, and increased risk of arterial and venous thrombotic disease. Our studies suggest that elevated leptin levels may directly promote arterial thrombosis in vivo. We found that leptin-deficient ob/ob mice had prolonged times to thrombosis after arterial injury with ferric chloride and that exogenously administered leptin corrected their phenotype in a dose-dependent manner. These effects appear to result from a direct, receptor-mediated effect of leptin on platelets, because leptin stimulated the aggregation of murine (wild-type and ob/ob) and human platelets, but it had no effect on platelets from leptin receptor-deficient db/db mice. Moreover, db/db mice had an attenuated thrombotic response to ferric chloride injury (indistinguishable from that of the ob/ob mice), which was unaffected by exogenous leptin. Our results raise the possibility that elevated plasma levels of leptin may contribute to the risk of atherothrombotic complications in human obesity.

Expression of transforming growth factor-beta and tumor necrosis factor-alpha in the plasma and tissues of mice with lupus nephritis.

Although elevated levels of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) have been implicated in renal disease, the tissue distribution and cellular localization of the induced cytokines is not well established. In this study, we investigated the expression of these cytokines during the progression of lupus nephritis in MRL lpr/lpr mice. The concentration of both cytokines increased in the plasma of these animals in an age-dependent manner, and there was an age-dependent induction of TGF-beta and TNF-alpha mRNAs in their kidneys. Although the increase in TGF-beta mRNA was specific for the kidney, the increase in TNF-alpha mRNA was widespread and also could be demonstrated in the liver, lung, and heart. In situ hybridization analysis of renal tissues from the lupus-prone mice localized TGF-beta mRNA to the glomerulus, and more specifically, to resident glomerular cells and inflammatory cells infiltrating periglomerular spaces in the nephritic lesions. The signals for TNF-alpha mRNA were detected only in inflammatory cells and were distributed throughout the nephritic kidney. Plasminogen activator inhibitor-1 (PAI-1) is known to be elevated in the glomeruli of MRL lpr/lpr mice, and intraperitoneal administration of either TGF-beta or TNF-alpha into normal mice markedly induced the expression of this potent inhibitor of fibrinolysis in renal glomerular or tubular cells in vivo. These results suggest that the increased renal expression of both cytokines may contribute to the development of lupus nephritis in this model and raise the possibility that PAI-1 may be involved. The fact that TGF-beta is specifically induced in the kidney whereas TNF-alpha increases in a variety of tissues, supports the hypothesis that the renal specificity of this disorder reflects the abnormal expression of TGF-beta.

Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease.

The fat mouse. A powerful genetic model to study hemostatic gene expression in obesity/NIDDM.

In this chapter, we summarize our studies on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and transforming growth factor beta (TGF-beta) expression in obesity, using genetically obese mice as a model. These studies emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also implicate multiple cytokines, hormones, and growth factors in the abnormal expression of these and perhaps other hemostatic genes by adipocytes in obesity/NIDDM. These studies demonstrate that tumor necrosis factor alpha (TNF-alpha) plays a central role in the expression of hemostatic genes in this disorder.

Glomerular extracellular matrix accumulation in experimental anti-GBM Ab glomerulonephritis.

Thickening of the glomerular basement membrane (GBM) results from excessive accumulation of extracellular matrix (ECM) proteins following glomerular injury. We studied the temporal relationship between the expression of growth factors, ECM accumulation, ECM degrading proteinases, and their inhibitors in a rat model of anti-GBM antibody (Ab) glomerulonephritis (GN) by the RNase protection assay and immunohistochemistry. There were two- or fourfold increases in the expression of transforming growth factor-beta(1) (TGF-beta(1)) and platelet-derived growth factor (PDGF) A and B chain mRNAs 4 days after anti-GBM Ab administration. These changes were temporally associated with increased accumulation of alpha1(III) and alpha2(IV) collagens, fibronectin, and heparan sulfate proteoglycan along the GBM. The increase in matrix accumulation was associated with little or no increases in the proteinases, urokinase plasminogen activator (u-PA) and transin, respectively. There was a 1.6x increase in the u-PA/28s mRNA ratio on day 4 in rats with anti-GBM Ab GN, but this was not associated with an increase in u-PA biologic activity. By comparison, the mRNAs of the proteinase inhibitors, plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase (TIMP) were 5x greater than that of control rats on day 4. PAI-1 mRNA correlate with increased biologic activity. These data demonstrate a temporal association between TGF-beta(1) and PDGF expression and matrix accumulation within the GBM in anti-GBM Ab GN. In addition, it suggest that this matrix accumulation results from an imbalance between matrix synthesis and degradation.

Regulation of murine protein C gene expression in vivo: effects of tumor necrosis factor-alpha, interleukin-1, and transforming growth factor-beta.

Protein C is a precursor of the anticoagulant serine protease, activated protein C, which inhibits coagulation factors Va and VIIIa. Although the liver appears to be the primary site of protein C synthesis, we previously demonstrated that the kidney and male reproductive organs also expressed abundant protein C mRNA in the mouse. In the present study, we further investigated the effects of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and transforming growth factor-beta (TGF-beta) on the expression of protein C mRNA in the principal producing organs, i.e., the liver, kidney, and testis. Both quantitative reverse transcription-PCR assay and in situ hybridization analysis revealed that TNF-alpha decreased protein C mRNA expression in the liver, kidney, and testis. IL-1 also down-regulated protein C mRNA expression in the liver and testis, but not in the kidney. In contrast, TGF-beta unchanged the expression level of protein C mRNA in these three organs. These observations suggest that TNF-alpha and IL-1 may contribute to an increase in the procoagulant potential by downregulation of protein C synthesis in the tissues during inflammatory processes.