The role of the platelet pool of Plasminogen Activator Inhibitor-1 in well-controlled type 2 diabetes patients.

BACKGROUND: The main inhibitor of the fibrinolytic system, Plasminogen Activator Inhibitor -1 (PAI-1), irreversibly binds tissue-type Plasminogen Activator (t-PA) and thereby inhibits the protective action of tPA against thrombus formation. Elevated levels of plasma PAI-1 are associated with an increased risk of cardiovascular events and are observed in subjects with type 2 diabetes (T2D) and obesity. Platelets contain the majority of PAI-1 present in blood and exhibit the ability to synthesis active PAI-1. Diabetic platelets are known to be hyper-reactive and larger in size; however, whether these features affect their contribution to the elevated levels of plasma PAI-1 in T2D is not established. OBJECTIVES: To characterize the PAI-1 antigen content and the mRNA expression in platelets from T2D subjects compared to obese and lean control subjects, in order to elucidate the role of platelet PAI-1 in T2D. METHODS: Nine subjects with T2D and obesity were recruited from Primary Care Centers together with 15 healthy control subjects (8 lean subjects and 7 with obesity). PAI-1 antigen levels in plasma, serum and platelets were determined by ELISA, and PAI-1 mRNA expression was analyzed by qPCR. RESULTS: There was no significant difference in PAI-1 mRNA expression or PAI-1 antigen in platelets in T2D subject in comparison to obese and lean control subjects. An elevated level of plasma PAI-1 was seen in both T2D and obese subjects. PAI-1 gene expression was significantly higher in both obese groups compared to lean. CONCLUSION: Similar levels of protein and mRNA expression of PAI-1 in platelets from T2D, obese and lean subjects indicate a limited role of platelets for the elevated plasma PAI-1 levels. However, an increased synthesis rate of mRNA transcripts in platelets from T2D and an increased release of PAI-1 could also result in similar mRNA and protein levels. Hence, synthesis and release rates of PAI-1 from platelets in T2D and obesity need to be investigated to further elucidate the role of platelets in obesity and T2D.

Hyperinsulinemia and insulin resistance in the obese may develop as part of a homeostatic response to elevated free fatty acids: A mechanistic case-control and a population-based cohort study.

BACKGROUND: It is commonly accepted that in obesity free fatty acids (FFA) cause insulin resistance and hyperglycemia, which drives hyperinsulinemia. However, hyperinsulinemia is observed in subjects with normoglycaemia and thus the paradigm above should be reevaluated. METHODS: We describe two studies: MD-Lipolysis, a case control study investigating the mechanisms of obesity-driven insulin resistance by a systemic metabolic analysis, measurements of adipose tissue lipolysis by microdialysis, and adipose tissue genomics; and POEM, a cohort study used for validating differences in circulating metabolites in relation to adiposity and insulin resistance observed in the MD-Lipolysis study. FINDINGS: In insulin-resistant obese with normal glycaemia from the MD-Lipolysis study, hyperinsulinemia was associated with elevated FFA. Lipolysis, assessed by glycerol release per adipose tissue mass or adipocyte surface, was similar between obese and lean individuals. Adipose tissue from obese subjects showed reduced expression of genes mediating catecholamine-driven lipolysis, lipid storage, and increased expression of genes driving hyperplastic growth. In the POEM study, FFA levels were specifically elevated in obese-overweight subjects with normal fasting glucose and high fasting levels of insulin and C-peptide. INTERPRETATION: In obese subjects with normal glycaemia elevated circulating levels of FFA at fasting are the major metabolic derangement candidate driving fasting hyperinsulinemia. Elevated FFA in obese with normal glycaemia were better explained by increased fat mass rather than by adipose tissue insulin resistance. These results support the idea that hyperinsulinemia and insulin resistance may develop as part of a homeostatic adaptive response to increased adiposity and FFA. FUNDING: Swedish-Research-Council (2016-02660); Diabetesfonden (DIA2017-250; DIA2018-384; DIA2020-564); Novo-Nordisk-Foundation (NNF17OC0027458; NNF19OC0057174); Cancerfonden (CAN2017/472; 200840PjF); Swedish-ALF-agreement (2018-74560).

Differential response of plasma plasminogen activator inhibitor 1 after weight loss surgery in patients with or without type 2 diabetes.

BACKGROUND: Obesity and type 2 diabetes (T2D) are associated with a suppression of fibrinolysis and an increased risk of intravascular thrombi because of elevated plasma plasminogen activator inhibitor 1 (PAI-1). OBJECTIVES: The aim was to investigate PAI-1 levels in obese patients in the early phase after bariatric surgery, before any weight loss, and in the late phase, to identify the impact of reduced adipose mass versus weight loss independent effects on PAI-1 levels. We also studied the impact of T2D on the rate of PAI-1 reduction. SETTINGS: Twelve obese patients with and without T2D (n = 6) who were scheduled for surgery at a designated Center of Excellence. METHODS: Plasma PAI-1 antigen was measured by enzyme-linked immunosorbent assay (ELISA) preoperatively and at 4 and 42 days after gastric bypass surgery. RESULTS: In the early phase, plasma PAI-1 was significantly decreased by 53% (P = .023). This difference did not remain significant in the late phase. However, PAI-1 levels in T2D and non-T2D patients were significantly different (P = .005). In non-T2D patients, plasma PAI-1 levels decreased significantly in both early and late phases (P = .038). Interestingly, in the T2D group, the PAI-1 levels tended to increase in the late phase and differed significantly from the non-T2D group. CONCLUSION: We report decreased PAI-1 levels in the immediate postoperative period after gastric bypass, indicating that a mechanism not related to the fat mass regulates the PAI-1 levels. Additionally, there may be a difference in PAI-1 levels between T2D and non-T2D patients 42 days postoperatively. Further studies are required to verify this difference and to elucidate the specific mechanisms responsible for PAI-1 synthesis.

Normalization of qPCR in platelets - YWHAE a potential genericreference gene.

The mRNA of human platelets has been extensively studied and it is generally appreciated that platelets contain mRNA transcripts derived from the megakaryocytes, and they have the ability to translate it into proteins. Additionally, platelets contain microRNA (miRNA) that has been shown to potentially regulate the translation of certain proteins. When quantifying gene expression by quantitative real-time polymerase chain reaction (qPCR), a valid normalization method is required and the use of reference genes is a common and robust approach. It is recommended to perform a proper validation of potential reference genes for each individual experimental setup. Previous studies have mainly been performed using commonly used reference genes for nucleated cells, and to our knowledge there are no global evaluations of the stability of transcripts in platelets. Finding a stable transcript would be valuable for inter-study comparisons, and the aim of this study was to identify one or more stable mRNA transcripts suitable as generic reference genes for mRNA gene expression studies in platelets. Platelets were incubated for 24 h and microarray of platelet mRNA revealed that the levels of YWHAE, B2M, ITM2B, H3F3A, PF4V1 remained similar between 0 and 24 h. Further validation of the stability of these genes together with GAPDH, RN18S1, and PPIA, genes frequently used as reference genes in platelet studies, was performed using qPCR after different in vitro conditions. In addition, inter-individual stability of the genes was analyzed in diabetic patients compared with healthy matched controls. Analysis of gene stability by the software RefFinder revealed that YWHAE, PF4V1, and B2M were the most stable genes in platelets from healthy donors. In addition, YWHAE was stable between subjects. Furthermore, the potential influence of miRNA on the selected genes was investigated by knockdown of Dicer1 in the megakaryocytic cell line MEG01. YWHAE, H3F3A, B2M, and GAPDH remained unchanged over time in MEG01 cells indicating that these genes are not regulated by miRNA and hence are more stably expressed. In conclusion, YWHAE is a stable transcript in platelets and we suggest the use of YWHAE as a generic reference gene in mRNA gene expression studies.

Platelets retain high levels of active plasminogen activator inhibitor 1.

The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125)I, and (125)I-tPA and (125)I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.

Mild hypothermia markedly reduces ischemia related coronary t-PA release.

In experimentally induced myocardial ischemia, mild hypothermia (33-35 degrees C) has a robust cardioprotective effect. Tissue plasminogen activator (t-PA) is a profibrinolytic enzyme that is released from the vascular endothelial cells in response to ischemia and other injurious stimuli. t-PA has also been found to have proinflammatory properties that could contribute to reperfusion injury. We postulated that hypothermia could attenuate t-PA release in the setting of myocardial ischemia. Sixteen 25-30 kg pigs were anesthetized and a temperature of 37 degrees C was established using an intravascular cooling/warming catheter. The pigs were then randomized to hypothermia (34 degrees C) or control (37 degrees C). A doppler flow wire was placed distal to a percutaneous coronary intervention balloon positioned immediately distal to the first diagonal branch of the left anterior descending artery (LAD). The LAD was then occluded for 10 min in all pigs. Coronary blood flow and t-PA was measured before, during and after ischemia/reperfusion. t-PA was measured in peripheral arterial blood and locally in the venous blood from the coronary sinus. Net t-PA release over the coronary bed was calculated by subtraction of arterial values from coronary sinus values. An estimate of differences in total t-PA release was calculated by multiplying net t-PA release with the relative increase in flow compared to baseline, measured in relative units consisting of ((ng/ml - ng/ml) x (cm/s/cm/s)). There was no observed difference in t-PA levels in peripheral arterial samples. As shown previously, net t-PA release increased during reperfusion. Hypothermia significantly inhibited the increase in t-PA release during reperfusion (peak value 9.44 +/- 4.34 ng/ml vs. 0.79 +/- 0.45 ng/ml, P = 0.02). The effect was even more prominent when an estimation of total t-PA release was performed with mean peak value in the control group 26-fold higher than in the hypothermia group (69.74 +/- 33.86 units vs. 2.62 +/- 1.10 units, P = 0.01). Mild hypothermia markedly reduces ischemia related coronary tissue plasminogen activator release. The reduction of t-PA release may contribute to the cardioprotective effect of hypothermia.

Heterogeneous glycosylation patterns of human PAI-1 may reveal its cellular origin.

The main inhibitor of intravascular fibrinolysis is plasminogen activator inhibitor 1 (PAI-1) which binds to and irreversibly inhibits tissue plasminogen activator (tPA). PAI-1 is present in blood, both in platelets and in plasma, and PAI-1 levels are associated with risk of atherothrombosis. Several tissues express PAI-1 but the source of plasma PAI-1 is not known. We recently found that platelets can de novo synthesize PAI-1 and the amount synthesized in vitro in 24 hours is 35-fold higher than required to maintain normal plasma levels. Recombinant human PAI-1 expressed in different cell types or secreted naturally by human cell lines, exhibit heterogeneous glycosylation patterns. The aim of this study was to investigate the hypothesis that platelets might be the source of plasma PAI-1 and that the cellular source of PAI-1 can be determined by its tissue-specific glycosylation pattern. PAI-1 was isolated from platelets, macrophages, endothelial cells, adipose tissue, as well as plasma from lean and obese subjects. The glycosylation was analyzed by nanoLC-MS/MS. PAI-1 isolated from cell lysates and conditioned media from macrophages, endothelial cells, and adipose tissue expressed heterogeneous glycosylation patterns. By contrast, no glycans were detected on PAI-1 isolated from plasma or platelets from healthy lean individuals. Hence, our data suggest that platelets may be the main source of plasma PAI-1 in lean individuals. Interestingly, plasma PAI-1 from obese subjects had a glycan composition similar to that of adipose tissue suggesting that obese subjects with elevated PAI-1 levels may have a major contribution from other tissues.

Plasminogen activator inhibitor 1 expression in platelets is not influenced by the 4G/5G promoter polymorphism.

In the present study we investigated the influence of the 4G/5G promoter polymorphism of the PAI-1 gene on the levels of PAI-1 mRNA and protein in platelets. After a screening of healthy male subjects, thirty-eight subjects homozygote for either the 4G or 5G allele were investigated. mRNA levels were quantified by real-time PCR and PAI-1 antigen in platelets and plasma was analysed by ELISA. The platelet PAI-1 mRNA levels correlated significantly with the PAI-1 antigen content, but there was no association between the polymorphism and mRNA levels, or protein levels in platelets. Also, plasma levels of PAI-1 antigen were not associated with homozygosity of the 4G/5G polymorphism, but as expected BMI and triglycerides emerged as significant predictors of plasma PAI-1 levels. The importance of the 4G/5G polymorphism on PAI-1 levels is controversial and the present study shows that although levels of platelet mRNA are related to its content of PAI-1 protein, there is no association between the 4G/5G promoter polymorphism and platelet PAI-1 mRNA or protein expression.

Platelets synthesize large amounts of active plasminogen activator inhibitor 1.

Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P = .001). Metabolic radiolabeling with (35)S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). This functional assay showed that the majority of the new protein was in an active configuration and could complex-bind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots.