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Division of Endocrinology/Diabetes/Metabolism and the Clinical Research Center (G.B., C.H., P.C.), Division of Hematology and the Sol Sherry Thrombosis Research Center (V.R.V., A.K.R.), Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Address all correspondence and requests for reprints to: Guenther Boden, M.D., Temple University Hospital, 3401 North Broad Street, Philadelphia, Pennsylvania 19140. E-mail: bodengh{at}tuhs.temple.edu; or A. Koneti Rao, M.D., Temple University School of Medicine, Sol Sherry Thrombosis Research Center, 3400 North Broad Street, Philadelphia, Pennsylvania 19140. E-mail: koneti{at}temple.edu.
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Abstract |
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Objective: Our objective was to examine the effects of hyperglycemia and hyperinsulinemia on the TF pathway of blood coagulation in T2DM.
Design: Three study protocols were used: 1) acute correction of hyperglycemia (with iv insulin) followed by 24 h of euglycemia, 2) 24 h of selective hyperinsulinemia, and 3) 24 h of combined hyperinsulinemia and hyperglycemia.
Setting: The study took place at a clinical research center.
Study Participants: Participants included 18 T2DM patients and 22 nondiabetic controls.
Results: Basal TF-procoagulant activity (TF-PCA), monocyte TF mRNA, plasma coagulation factor VII (FVIIc), and thrombin-anti-thrombin complexes were higher in T2DM than in nondiabetic controls, indicating a chronic procoagulant state. Acutely normalizing hyperglycemia over 2–4 h resulted in a small (
7%) but significant decline in TF-PCA with no further decline over 24 h. Raising insulin levels alone raised TF-PCA by 30%, whereas raising insulin and glucose levels together increased TF-PCA (by 80%), thrombin-anti-thrombin complexes, and prothrombin fragment 1.2. Plasma FVIIa and FVIIc declined with increases in TF-PCA.
Conclusion: We conclude that the combination of hyperglycemia and hyperinsulinemia, common in poorly controlled patients with T2DM, contributes to a procoagulant state that may predispose these patients to acute cardiovascular events.
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Introduction |
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Patients with T2DM have elevated levels of circulating TF-procoagulant activity (TF-PCA) (15, 16). We have recently shown in nondiabetic volunteers that raising blood insulin levels and especially raising blood glucose and insulin together to levels frequently seen in patients with T2DM increased circulating TF-PCA and monocyte TF expression and lowered plasma FVIIa and FVIIc (17, 18). These findings suggested that the chronically elevated TF-PCA in T2DM may be, at least in part, caused by their hyperglycemia-hyperinsulinemia. To test this hypothesis, we have investigated in patients with T2DM (and in nondiabetic controls) the effects of changes in glucose and insulin levels on circulating TF-PCA, on monocyte TF expression, and on plasma coagulation factors VII (FVIIc), its activated form (FVIIa) and FVIII, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1.2 (F1.2), both of which are sensitive indicators of thrombin generation.
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Subjects and Methods |
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Eighteen patients with T2DM participated in the three different studies (Table 1
). One patient was treated with sulfonylureas, five with metformin, two with insulin, and one with diet alone; three were treated with sulfonylureas plus metformin, three with insulin plus metformin, two with sulfonylureas, thiazolidinediones, and metformin, and one with insulin, thiazolidinediones, and metformin. These medications were withheld at least 72 h before hospital admissions. Twenty-two nondiabetic volunteers underwent the same three studies as the patients with T2DM and served as controls. Results from the control studies, which were done 1–5 months before the T2DM studies, have been reported previously (17). Informed written consent was obtained from all subjects after explanation of the nature, purpose, and potential risks of the studies. The study protocol was approved by the Institutional Review Board of Temple University Hospital.
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All studies began between 0600 and 0800 h after an overnight fast with the subjects reclining in bed. Subjects were fasting but were allowed to drink water ad libitum and remained at bed rest for the duration of the study. A short polyethylene catheter was inserted into an antecubital vein for infusion of test substances. Another catheter was placed into the contralateral forearm vein for blood sampling. This arm was wrapped with a heating blanket (70 C) to arterialize venous blood. Blood was collected for determination of basal circulating TF-PCA, plasma FVIIa, FVIIc, FVIII, TAT, F1.2, and TF pathway inhibitor (TFPI) levels, and monocyte TF mRNA. After that, the following studies were performed.
Correction of hyperglycemia (basal glucose/basal insulin clamps). The purpose of these studies was to examine effects of lowering of blood glucose on TF-PCA and other coagulation parameters. Starting at 0600 h, blood glucose was normalized over a 2- to 4-h period with iv infusion of regular insulin. After that, normal plasma glucose levels were maintained for 24 h by iv infusion of small amounts of insulin (0.04–1.1 U/h) when needed.
Selective elevation of insulin (basal glucose/high insulin clamps). The purpose of these studies was to examine effects of changing serum insulin levels on TF-PCA and other coagulation parameters. Lowering of the elevated serum insulin levels in these patients was not possible because this could have been accomplished only with somatostatin, which itself elevates TF-PCA levels (19). Regular human insulin (Humulin; Eli Lilly, Indianapolis, IN) was infused iv at a rate of 12 pmol/kg·min. Glucose was maintained at approximately 5.5 mmol/liter (100 mg/dl) by feedback-controlled glucose infusions. Glucose concentrations were determined in 0.25-ml blood samples every 15–30 min or more frequently at the beginning and at 1- to 2-h intervals later with a glucose analyzer, and the glucose infusion rates were adjusted as needed. Plasma electrolytes were monitored every 6 h, body weight every 12 h, and fluid balances every 6 h. Potassium (20 mg) and magnesium (400 mg) were given orally every 12 h.
Combined elevation of glucose and insulin (high glucose/high insulin clamps).
The purpose of these studies was to examine effects of raising blood glucose and insulin levels on TF-PCA and other coagulation-related events. Again, because of the independent effects of somatostatin on TF-PCA, lowering of insulin and glucose levels together was not feasible. A 20% glucose solution was infused iv at variable rates, which were adjusted to maintain plasma glucose at approximately 200 mg/dl (11 mmol/liter). Small blood samples (0.25 ml) were collected every 30–60 min initially and every 1–2 h later for measurement of blood glucose concentrations. Plasma electrolyte and fluid balances were monitored as described above.
Assays
Blood samples were collected from antecubital veins without tourniquet-induced venostasis at 0, 6, 12, 18, and 24 h. Plasma glucose was measured with a glucose analyzer using the glucose oxidase method and serum insulin by RIA using an antiserum with minimal (<0.2%) cross-reactivity with proinsulin (Linco Research, St. Charles, MO). Serum triglycerides were measured enzymatically. Electrolytes, total cholesterol, and high density lipoprotein were measured at the Temple University Hospital Chemistry Laboratory.
TF-PCA was measured in whole-blood cell lysates with a two-stage clotting assay using recombinant FVIIa (American Diagnostica, Greenwich, CT), factor X (Enzyme Research Laboratories, South Bend, IN), and normal human plasma containing phospholipids vesicles, as described previously (13, 17). This assay measures cell-bound and microparticle-associated TF in lysed membranes obtained from whole blood.
Plasma FVIIa, FVIIc, and FVIII activities were measured with clotting assays as described previously (17). Thrombin generation was assessed by determination of F1.2 and TAT complexes in plasma using ELISA (Enzygnost; Dade Behring, Marburg, Germany). TFPI antigen levels were measured with the Imubind total TFPI ELISA from American Diagnostica.
TF-mRNA in monocytes was determined by real-time RT-PCR analysis as described previously (17).
Statistical analysis
A one-way ANOVA was used to test for significant differences between studies with Student-Newman-Keuls post hoc analysis. If data were not normally distributed, the Kruskal-Wallis one-way ANOVA with Dunn’s post hoc analysis was used. To test for differences across time, a one-way repeated-measures ANOVA with Student-Newman-Keuls post hoc analysis was used. If data were not normally distributed, the Friedman repeated-measures ANOVA on ranks with Student-Newman-Keuls post hoc analysis was used. Statistical differences between d 1 (baseline) and d 2 (24 h) were determined with a paired t test and, if not normally distributed, with the Wilcoxon signed rank test. Statistical analyses were performed using SigmaStat for Windows (version 2.0; SPSS, Chicago, IL). Statistical significance was defined as P < 0.05. All results are presented as means ± SE.
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Results |
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)
Basal TF-PCA (69.5 ± 2.8 vs. 17.5 ± 0.9 U/ml, P < 0.001), monocyte TF/18s mRNA ratios (9.2 ± 1.1 x 10–4 vs. 6.0 ± 0.7 x 10–4, P < 0.02), and plasma FVIIc (1.23 ± 0.06 vs. 0.93 ± 0.04 U/ml, P < 0.001), FVIII (1.25 ± 0.15 vs. 0.94 ± 0.03 U/ml, P < 0.04), and TAT (11.8 ± 2.4 vs. 4.9 ± 0.6 µg/liter, P < 0.005) were all higher in patients with T2DM than in nondiabetic controls, whereas FVIIa [91 ± 8 vs. 78 ± 5 mU/ml, not significant (NS)] tended to be elevated. In our previous study with a larger number of T2DM patients, plasma FVIIa levels were significantly higher (16). The nondiabetic controls were younger (37 vs. 50 yr) and weighed less (87.5 vs. 106.5 kg) than the patients with T2DM (Table 1
). However, because neither body weight (body weight vs. TF-PCA, r = 0.31, P = 0.26) nor age (age vs. TF-PCA, r = 0.2, P = 0.4) correlated with TF-PCA, the differences in weight and age between the two groups could not explain the large differences in circulating TF-PCA.
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)To explore effects of acutely improving glycemic control, fasting plasma glucose was lowered (from 13.3 ± 2.3 to 5.3 ± 0.4 mmol/liter) with iv insulin within 2–4 h in six patients with T2DM. During this short period of rapidly increasing insulin and decreasing glucose, TF-PCA decreased by 7% (from 72 to 67 U/ml, P < 0.02) and FVIIa by 19% (from 91 to 74 mU/ml, P < 0.02), whereas FVIIc did not change significantly. Glucose was then kept at euglycemic levels for 24 h with iv infusion of insulin at low rates (0.04–1.10 U/h). TF-PCA did not decrease (67 ± 4 at 0 h vs. 65 ± 13 U/ml at 24 h, NS) during the 24 h of euglycemia and remained more than 3-fold higher than in nondiabetic controls. The monocyte 24 h/0 h TF/18s mRNA ratio did not decrease (1.06 ± 0.14, NS). Plasma FVIIc decreased (from 1.48 ± 0.11 to 1.12 ± 0.08 mU/ml, P < 0.03). There were no significant changes in plasma FVIIa activity (74 ± 11 vs. 67 ± 7 mU/ml, NS), FVIII (1.63 ± 0.4 vs. 1.26 ± 0.14 mU/ml, NS), TAT (9.6 vs. 7.2 µg/liter, NS), and F1.2 (0.6 vs. 1.1 nmol/liter, NS).
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)
To determine effects of selective hyperinsulinemia, serum insulin was increased by iv infusion of insulin (2 mU/kg·min, or 14 U/h). This raised serum insulin to a mean level of 962 ± 89 pmol/liter over 24 h in six patients with T2DM and to 1278 ± 124 pmol/liter in six controls (the difference between the two groups was not statistically significant). In the T2DM patients, plasma glucose decreased rapidly in response to the insulin infusion and thereafter was clamped in both groups at approximately 5.5 mmol/liter with variable-rate glucose infusions.
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The monocyte TF mRNA 24 h/0 h ratios increased to 1.5 ± 0.1 in T2DM (P < 0.04). Plasma VIIc declined from 1.14 and 0.88 U/ml at 0 h to 0.80 and 0.77 U/ml in patients and controls, respectively (P < 0.001 for T2DM, P < 0.05 for controls). The corresponding declines in FVIIa were from 78.2 and 83.2 to 52.1 and 44.7 U/ml, respectively (P < 0.001). TAT and F1.2 did not change significantly in T2DM but both rose in controls (TAT from 12.3 ± 8 µg/liter at 0 h to 25.1 ± 6 µg/liter at 18 h and F1.2 from 1.1 ± 0.1 nmol/liter at 0 h to 1.7 ± 0.3 nmol/liter at 18 h, both P < 0.05). TFPI (34.1 ± 2.3 ng/ml at 0 h vs. 30.5 ± 2.0 ng/ml at 24 h, NS) and FVIII (0.9 ± 0.1 vs. 1.1 ± 0.1 U/ml) did not change significantly.
Effects of raising both glucose and insulin (Fig. 4)
To determine effects of worsening glycemic control, plasma glucose levels were raised by iv glucose infusion to approximately 12 mM in both groups. (No insulin was infused.) This increased serum insulin levels from 135 ± 47 pmol/liter at 0 h to a mean of 356 ± 22 pmol/liter over 24 h in T2DM and from 71 ± 10 pmol/liter at 0 h to a mean of 857 ± 49 pmol/liter over 24 h in controls. The difference between mean 24-h insulin in T2DM and normal subjects was not statistically significant.
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The monocyte TF mRNA 24 h/0 h ratio increased from 1.0 to 1.21 ± 0.6 in T2DM (P < 0.01) and from 1.0 to 1.9 ± 0.3 in controls (P < 0.004).
FVIIc declined by about 50% and about 40%, respectively, in patients with T2DM and controls, whereas FVIIa declined by about 20% in both groups. FVIII increased in controls (0.99 to 1.26, P < A 0.001) but not in T2DM (1.22 to 1.16, NS).
TAT increased from 5.2 ± 1.6 µg/liter at 0 h to 31.7 ± 10.7 µg/liter at 24 h in T2DM (P < 0.001) and from 5.2 ± 1.9 to 33.7 ± 5.7 µg/liter in controls (P < 0.02). F1.2 increased from 0.8 ± 0.1 nmol/liter at 0 h to 2.7 ± 0.7 nmol/liter at 24 h in T2DM and from 1.0 ± 0.1 to 2.2 ± 0.3 nmol/liter in controls (both P < 0.05). TFPI did not change significantly (34.6 ± 3.5 at 0 h vs. 38.1 ± 4.2 ng/ml at 24 h, NS).
TF-PCA correlated positively with TAT (r = 0.47, P < 0.001) and with F1.2 (r = 0.45, P < 0.001) in controls and T2DM.
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Discussion |
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Confirming a preliminary report from our laboratory (16), and findings by others (15), we found that under basal conditions, TF-PCA was higher in patients with T2DM than in nondiabetic controls. We have now expanded these findings by showing that monocyte TF mRNA and plasma FVIIc and FVIII were also higher in patients with T2DM and that TAT levels were elevated, indicating enhanced thrombin generation. Together, these results demonstrated that these patients were in a chronic procoagulant state. This is clinically important because elevated TF-PCA and plasma FVII/VIIa have been associated with increased occurrence of arterial events (20, 21).
Monocytes are the only hematopoietic cells with established ability to synthesize TF-PCA (9, 10). The higher TF mRNA in monocytes of patients with T2DM suggested that the increase in TF-PCA was caused, at least in part, by an increase in TF transcription.
We have also shown that selectively raising insulin levels in patients with T2DM resulted in an approximately 30% increase in TF-PCA above the already high basal level (from 61 to 79 U/ml, P < 0.05) but had no demonstrable effect on TAT or F1.2. In normal controls, by comparison, the insulin induced a 6-fold increase in TF-PCA, presumably because of the lower basal level, and was associated with an increase in TAT at 18 h.
More impressive changes were seen when both insulin and glucose concentrations were raised together. For instance, in patients with T2DM, TF-PCA rose approximately two times higher than with selective hyperinsulinemia (151 vs. 79 U/ml, P < 0.001) and, importantly, was associated with significant increases in TAT and F1.2. Moreover, TF-mRNA in monocytes increased significantly. These results demonstrated that hyperglycemia potentiated the effects of hyperinsulinemia on the TF pathway of coagulation. Similar results were obtained in the nondiabetic controls (Fig. 4). These observations suggested that the high risk for thrombotic events in these patients may increase further when glycemic control deteriorates. Also, the increase in TF-PCA levels in response to selective hyperinsulinemia suggested that a rise in serum insulin, perhaps caused by an increase in insulin resistance, may also increase the risk for thrombotic events. Supporting this notion are numerous studies that have shown a positive relationship between insulin resistance and cardiovascular outcomes (22, 23, 24).
Patients with T2DM participating in the three 24-h studies were older than the nondiabetic controls, and those participating in the selective hyperinsulinemia study were also heavier (Table 1). However, neither TF-PCA nor FVIIc or TAT correlated significantly with age or weight in these studies (TF-PCA vs. age, P = 0.17, P = 0.09, and P = 0.60, respectively, in the 24-h euglycemia, selective hyperinsulinemia, and hyperglycemia plus hyperinsulinemia studies). Thus, the differences in age and weight could not explain the differences in circulating TF-PCA, FVIIc, and TAT (data not shown) observed during basal conditions (see Results) and during the three 24-h studies.
Several of our findings deserve comment. For instance, rapid lowering of plasma glucose over 2–4 h in T2DM patients was associated with a small but significant decline in TF-PCA (Fig. 2). That the decrease in glucose was the cause was supported by the finding that a similar decrease was also observed at 6 h in the selective hyperinsulinemia study (Fig. 3). We may have underestimated the effects of rapidly lowering glucose inasmuch as the decline in TF-PCA was probably diminished by the concurrent small increase in serum insulin. The lack of further declines during the subsequent 24-h period with euglycemia (Fig. 2) may suggest that lowering of blood glucose for longer periods are required for a further decline in TF-PCA. In fact, in another study, improved glycemic control over 3 months resulted in a significant decrease in circulating TF activity in patients with T2DM (16). Moreover, a part of the chronic elevation in TF-PCA may be due to underlying atherosclerosis and inflammation and may not be amenable to rapid decrease with correction of glucose alone. For example, patients with peripheral arterial disease have elevated TF-PCA, which is decreased by antiplatelet agents (25).
Effects of isolated hyperglycemia on TF-PCA are difficult to study because in nondiabetic subjects and T2DM patients, a rise in blood glucose invariably elevates serum insulin. When the rise in insulin was prevented in nondiabetic controls by infusion of somatostatin (pancreatic clamp), TF-PCA rose about 2-fold during 24 h of selective hyperglycemia in nondiabetic controls (17). Subsequent studies showed, however, that somatostatin per se increased basal TF-PCA about 2-fold and, more importantly, completely abolished the 9-fold increase in TF-PCA induced by hyperglycemia plus hyperinsulinemia (19). Presumably, therefore, any hyperglycemia-mediated increase in TF-PCA would also be abolished by somatostatin. Stegenga et al. (26) have recently reported that selective hyperglycemia for 6 h (induced using somatostatin) produced an approximately 2-fold increase in plasma TF antigen in healthy volunteers. In view of the effects of somatostatin on TF, their results are more likely the consequence of somatostatin than glucose. Thus, more studies are needed to clarify the effect of selective hyperglycemia on TF-PCA.
Another observation deserving comment was that plasma FVII and FVIIa activities were chronically elevated in the diabetic patients but decreased during our acute hyperinsulinemic and hyperglycemic experiments. Because TF is the principal ligand for FVIIa and FVIIc, we believe that the decrease in plasma FVII activity reflected an accelerated clearance from plasma as a result of its binding to an increased number of available TF binding sites on cells/membranes (18). Similar decreases in FVII have been reported after bolus infusions of endotoxin (27) and in sepsis (28), and an inverse relationship between plasma FVII and TF-PCA has been observed by us in other studies (17).
To explain why high insulin plus glucose promoted TF pathway activation more than high insulin alone, we considered the possibility that it may have been related to a difference in stimulated glucose uptake. This, however, appeared to be unlikely because the mean 24-h glucose uptake was more than two times higher in the high insulin compared with the high insulin plus glucose studies (7.2 ± 1.6 vs. 3.4 ± 1.1 mg/kg·min).
There is, however, an alternative and perhaps more likely mechanism.
Activated platelets have been shown to play a major role in decrypting and enhancing TF activity (29). We have previously shown in normal volunteers that combined hyperinsulinemia plus hyperglycemia strongly activated platelets (15, 18). Thus, it seems reasonable to propose that the large increase in TF pathway activity observed in response to hyperglycemia plus hyperinsulinemia may have been, at least partially, the result of platelet activation. In support, we have shown that platelet-inhibiting agents lower TF-PCA in patients with peripheral arterial disease (25).
Summary
Basal circulating TF-PCA, monocyte TF-mRNA, and plasma FVIIc, FVIII, and TAT were elevated in patients with T2DM, indicating the presence of a chronic procoagulant state. Rapidly normalizing glucose over 2–4 h resulted in a small but significant decrease in TF-PCA with persistently elevated levels throughout the 24-h period. Raising insulin for 24 h while keeping glucose at normal levels further increased TF-PCA. Raising glucose and insulin levels together resulted in a much larger increase of TF-PCA levels associated with larger increases in TAT and F1.2.
We conclude that a rise in plasma glucose and insulin, which is characteristically seen when glycemic control deteriorates in patients with T2DM, results in rapid and large increases in circulating TF-PCA associated with thrombin generation and thus is likely to increase the risk for acute cardiovascular events.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: V.R.V., P.C., and A.K.R. have nothing to disclose. G.B. received lecture fees from Novartis and Sanofi-Aventis, and C.H. received lecture fees from Pfizer, Inc.
First Published Online September 4, 2007
Abbreviations: F1.2, Prothrombin fragment 1.2; FVII, coagulation factor VII; NS, not significant; PCA, procoagulant activity; TAT, thrombin-antithrombin; T2DM, type 2 diabetes mellitus; TF, tissue factor; TFPI, TF pathway inhibitor.
Received April 24, 2007.
Accepted August 27, 2007.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, P < 0.001 compared with 0 h.