Crossing borders: the role of the endothelial glycocalyx and intravascular haemostasis in vascular complications of diabetes mellitus - Chapter 3: Glucose: a prothrombotic factor?

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UvA-DARE (Digital Academic Repository)

Crossing borders: the role of the endothelial glycocalyx and intravascular

haemostasis in vascular complications of diabetes mellitus

Lemkes, B.A.

Publication date

2011

Link to publication

Citation for published version (APA):

Lemkes, B. A. (2011). Crossing borders: the role of the endothelial glycocalyx and

intravascular haemostasis in vascular complications of diabetes mellitus.

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CHAPTER

3

Hyperglycaemia, a prothrombotic factor?

Bregtje A. Lemkes, Jeroen Hermanides, J. Hans DeVries, Frits Holleman, Joost C.M. Meijers and Joost B.L. Hoekstra

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Abstract

Diabetes mellitus is characterized by a high risk of atherothrombotic events. What is more, venous thrombosis has also been found to occur more frequently in this patient group. This prothrombotic condition in diabetes is underpinned by laboratory findings of elevated coagulation factors and impaired fibrinolysis. Hyperglycaemia plays an important role in the development of these haemostatic abnormalities, as is illustrated by the association with glycaemic control and the improvement upon treatment of hyperglycaemia. Interestingly, stress induced hyperglycaemia, which is often transient, has also been associated with poor outcome in thrombotic disease. Similar laboratory findings suggest a common effect of acute vs. chronic hyperglycaemia on the coagulation system. Many mechanisms have been proposed to explain this prothrombotic shift in hyperglycaemia, such as a direct effect on gene transcription of coagulation factors by hyperglycaemia-induced oxidative stress, loss of the endothelial glycocalyx layer, which harbours coagulation factors, as well as direct glycation of coagulation factors, altering their activity. In addition, both chronic and acute hyperglycaemia are often accompanied by hyperinsulinaemia, which has been shown to have prothrombotic effects as well. In conclusion, the laboratory evidence of the effects of both chronic and acute hyperglycaemia suggests a prothrombotic shift. Additionally, hyperglycaemia is associated with poor clinical outcome of thrombotic events. Whether intensive treatment of hyperglycaemia can prevent hypercoagulability and improve clinical outcome remains to be investigated.

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Patients with diabetes are notorious for their risk of vascular events. Apart from the effects of diabetes and its prerequisite hyperglycaemia on the development of atherosclerosis, this high risk may also be caused by the procoagulant state found in diabetes1, 2_.

In recent years hyperglycaemia per se, even without overt diabetes, has gained interest as a potential target to improve clinical outcomes in hospitalized patients with acute illness3_{. In this setting the effects of hyperglycaemia on the coagulation system may be}

of greater importance than previously considered. Here we discuss the current evidence regarding potentially harmful changes in the coagulation system and subsequent risk of thrombotic disease, not only caused by diabetes but also by acute hyperglycaemia.

Chronic hyperglycaemia

Type 2 diabetes

Type 2 diabetes (DM2) is defined by hyperglycaemia, but often accompanied by hyperinsulinaemia, dyslipidaemia, hypertension and obesity. Its effects on the coagulation system can therefore not easily be attributed to either one of these entities4_,

but the impact of glucose on coagulation in diabetes has been studied quite extensively.

Markers of fibrinolysis and coagulation

Both parameters of increased coagulability as well as a fibrinolytic impairment have been found in DM2, although there are many different markers in the circulation to measure these abnormalities. Platelet-dependent thrombin generation, for instance, was measured in patients with poor glycaemic control, good glycaemic control and healthy controls. In vitro induced thrombin generation was found to be increased in platelet-rich plasma from diabetes patients compared to healthy controls and a significant elevation of thrombin levels was also demonstrated in plasma from poorly controlled DM2 when compared to well controlled patients5_{. In a placebo-controlled trial using}

diet modification and troglitazone, a peroxisome proliferator-activated receptor (PPAR) γ agonist, a significant association was shown between improved glycaemic control and blood thrombogenicity as reflected by a reduction in ex-vivo thrombus formation in a Badimon perfusion chamber. Improved glycaemic control was the only significant predictor of a decrease in blood thrombogenicity irrespective of treatment allocation6_.

The contribution of hypercoagulability in DM2 to the development of cardiovascular disease was also illustrated by the increased levels of prothrombin fragment 1+2 (F1+2) found to be associated with the presence of proven cardiovascular disease in DM2 patients as compared to patients without cardiovascular disease7_.

In a group of poorly controlled DM2 patients (HbA1c 10%) extraordinarily high concentrations of plasminogen activator inhibitor-1 (PAI-1), indicating hypofibrinolysis, were detected leading the authors to conclude that profound hyperglycaemia is

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accompanied by profound increases in PAI-1. Subsequent treatment of hyperglycaemia by either glipizide or metformine as monotherapy comparably decreased PAI-1, which argues for an effect of glucose lowering rather than a specific medication effect8_{. This}

state of hypofibrinolysis is well established in DM2 and characterized by elevated levels of PAI-1 as well as prolonged clot lysis time7, 9-11_{. The impairment in the fibrinolytic system}

in DM2 is of interest since these impairments are independent primary risk factors for myocardial infarction12, 13_.

Hyperinsulinaemia

To disentangle the effects of glucose and insulin in type 2 diabetes, Boden et al studied the effects of acute correction of hyperglycaemia with insulin followed by either 24 hours of experimentally induced normo-insulinaemic euglycaemia, 24 hours of euglycaemic hyperinsulinaemia or 24 hours of combined hyperinsulinaemia and hyperglycaemia, in DM2 patients as well as healthy controls. They found baseline elevations of tissue factor procoagulant activity (TF-PCA), monocyte TF mRNA and plasma factor VII, factor VIII and thrombin-antithrombin (TAT) complexes in patients with DM2 compared with healthy controls. Normalizing glucose significantly decreased TF-PCA. Increasing insulin levels raised TF-PCA and elevating glucose and insulin levels together resulted in a much larger rise of TF-PCA, which was associated with increases in TAT and F1+214_{. Thus glucose and insulin both seem to play a role in the pathogenesis of the}

prothrombotic state in type 2 diabetes.

Effect of glucose lowering therapies

The effects of improved glycaemic control on PAI-1 levels in DM2 have been demonstrated for different oral antidiabetic therapies, such as metformin alone15_{or in combination}

with pioglitazone or rosiglitazone16_{and glimepiride in combination with pioglitazone}

or rosiglitazone17_{. Metformin, which already had proven beneficial cardiovascular}

effects in the United Kingdom Prospective Diabetes Study trial, also reduced factor VII and fibrinogen levels and shortened clot lysis time18_{. When metformin was added}

to a sulphonylurea derivative in poorly controlled elderly DM2 patients, the resulting substantial improvement in glycaemic control was accompanied by beneficial changes in markers of platelet function (platelet factor 4 and beta-thromboglobulin), thrombin generation (fibrinopeptide A, F1+2, and D-dimer) and fibrinolysis (PAI-1 activity and antigen)19_.

In the Diabetes Prevention Programme, which studied the stages preceding diabetes (i.e. impaired glucose tolerance) lifestyle interventions even more than metformin treatment showed a modest, but significant, amelioration in fibrinogen levels20_{. Data on the effects}

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conflicting than for the oral antidiabetic treatments. Although some authors report beneficial effects of insulin21_{, others were unable to find improvements with insulin}

therapy22, 23_. Type 1 Diabetes

In type 1 diabetes (DM1) patients the specific contribution of hyperglycaemia to the prothrombotic state is clearer as they lack the other risk factors which confound the relationship in DM2 patients. In a long term follow up study, a highly significant correlation was found between mean HbA1c in DM1 patients over the course of 18 years and impaired fibrinolysis as represented by elevated PAI-1 and decreased tissue plasminogen activator (t-PA)24_{. In a smaller setting, eight DM1 patients on continuous}

subcutaneous insulin infusion therapy had treatment withheld for the duration of four hours which caused a rise of PAI-1 and plasma TF. Although the authors conclude that early ketogenesis causes a prothrombotic change in DM1 patients, the effects of acute hyperglycaemia in this setting cannot be excluded25_{. Platelet function tests, including}

aggregation and platelet adhesion tests, did not improve with intensive glycaemic control in DM1. However, platelet function tests are notoriously variable and the authors may not have included a sufficient number of patients to overcome this disadvantage26_.

Finally, despite the abundant evidence of fibrinolytic impairment in diabetes, not all markers of fibrinolysis are abnormal. Thrombin-activatable fibrinolysis inhibitor (TAFI) for instance, showed no difference between patients with DM1 and healthy controls27_a

finding which was recently confirmed in DM2 patients7_. Diabetes and thrombosis

DM2 and, maybe to a lesser extent, DM1 are known for a high risk of developing atherothrombotic events. This is at least partly explained by hyperglycaemia, given the continuous relationship between the development of cardiovascular disease and glycaemic control, also in DM228_{. Moreover, intensive blood glucose control in the}

early stages of the disease proved effective in lowering the long term incidence of cardiovascular disease in both disease entities29, 30_.

Recently it has become clear that not only atherothrombotic events are seen more often in patients with diabetes but that venous thromboembolism (VTE) may also be more frequent in this patient group. Movahed et al.31_{found an odds ratio (OR) of 1.27 (95% CI}

1.19-1.35) for the occurrence of pulmonary embolism in DM patients. A few years before, Tsai et al 32_{also found diabetes to be a risk factor for VTE with a hazard ratio (HR) of 1.46}

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However, in the earlier Nurses’ Health Study such an association could not be found33_,

nor could Jones and Mitchell34_{find it in a smaller scale study in 1983. These apparently}

conflicting data on the effects of diabetes on the risk of venous thromboembolism have recently been subjected to meta-analysis, showing DM to be an independent risk factor for VTE with an OR of 1.41 (95%CI 1.12-1.77)35_{. Although the effect of glucose lowering}

on VTE risk remains to be established, the overrepresentation of both venous as well as atherothrombosis in diabetes is suggestive of a prothrombotic effect of its main component, hyperglycaemia, in addition to its more established effects on atherosclerosis.

Acute hyperglycaemia

Apart from chronic hyperglycaemia, it is important to consider the role of acute hyperglycaemia. Frequently, this is transient hyperglycaemia resulting from metabolic deterioration during (severe) illness36_{. Although this may result from pre-existing and}

undiagnosed diabetes, 30-40% of patients with ‘stress-hyperglycaemia’ will revert to normoglycaemia with follow-up37, 38_{. Transient hyperglycaemia will usually be}

accompanied by transient hyperinsulinaemia.

The effect of hyperglycaemia and hyperinsulinaemia on the coagulation system in subjects without diabetes has also been studied rather extensively. Already in 1988, Ceriello et al. 39_{showed that experimentally increased glucose levels activated the}

coagulation system in non-diabetic subjects by increasing factor VII clotting activity. Stegenga et al.40_{demonstrated in healthy volunteers that hyperglycaemia (12 mmol/l),}

irrespective of insulin levels, activates coagulation, marked by an increase in TAT complexes and soluble tissue factor (sTF). In contrast, hyperinsulinaemia inhibited fibrinolysis by increasing PAI-1 levels. This was even more profound when systemic inflammation was induced41_{. In vitro, studies with endothelial cells from pig aortas}

exposed to increasing glucose concentrations indicated that PAI-1 secretion and synthesis increased in parallel to glucose levels42_{. Activation of the tissue factor pathway following}

induction of hyperglycaemia in healthy volunteers was also observed in by Rao et al.43_{. In}

a subsequent study, 29 healthy volunteers were exposed to combinations of euglycaemia or hyperglycaemia with normoinsulinaemia or hyperinsulinaemia44_{. They found that}

selective hyperglycaemia and hyperinsulinaemia activated the coagulation system, but the combination of both showed the largest increase in sTF procoagulant activity, TF expression on monocytes and TF mRNA in monocytes, TAT, factor VII, factor VIII and platelet activation, measured by platelet expression of soluble CD40 ligand. Finally, Nieuwdorp et al.45_{discovered that hyperglycaemia in healthy volunteers concomitantly}

reduced the protective glycocalyx of the endothelium and the function of the endothelium itself and increased prothrombin fragment 1+2 and D-dimer levels.

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Acute hyperglycaemia and coagulation in the ICU

Two studies have attempted to elucidate the role of the coagulation system in relation to strict glycaemic control. In the first Leuven trial, van den Berghe successfully implemented strict glycaemic control in the ICU, showing a clear mortality benefit3_{. She}

published a sub-analysis, investigating the effect of strict glucose control on coagulation and fibrinolysis46_{. Although a variety of parameters was assessed, no differences were}

found between the intensively treated group and the control group. However, samples were obtained only at 5 and 10 days after admission and an acute effect within the first 5 days could have been missed. Savioli et al.47_{investigated the effect of strict glucose control}

on coagulation and fibrinolysis in patients with septic shock on admission and up to 28 days after admission. They found that strict glucose control reduced the impairment of the fibrinolytic system, as measured by PAI-1. However, strict glucose control in the ICU is now being heavily debated because of the recently published NICE-SUGAR trial, which showed increased mortality in the intervention group48_.

Acute hyperglycaemia and thrombosis

Several thrombotic conditions have been described to be accompanied by acute hyperglycaemia, most importantly myocardial infarction (MI), stroke and venous thromboembolism (VTE). What is more, clinical outcome may be influenced by hyperglycaemia even in absence of diabetes. During MI, for instance, hyperglycaemia on admission predicts morbidity and mortality in patients without previously diagnosed diabetes49-53_{. Furthermore, elevated glucose levels on admission are directly related to}

the infarct size and reductions in coronary flow after stent implantation in non-diabetic patients54, 55_.

This might be related to intravascular thrombotic events. Patients with acute coronary syndrome and admission glucose > 7.0 mmol/l had elevated values of thrombin-antithrombin complexes and platelets activation, measured by soluble CD40 ligand levels, as compared with patients admitted with glucose < 7.0 mmol/l56_{. Also the fibrin clot lysis}

time was impaired in hyperglycaemic subjects. In another study activation of platelets, as measured by beta-thromboglobulin, was associated with hyperglycaemia after MI, independent of pre-existing diabetes57_{. In a rabbit model, reducing hyperglycaemia}

using acarbose resulted in decreased infarct size58_.

In stroke patients, admission hyperglycaemia was related to the infarct size59-61_{and a}

strong predictor of post-stroke morbidity and mortality62, 63_{. Ribo et al.}64_{showed that}

acute but not chronic hyperglycaemia during stroke was associated with lower tissue-type plasminogen activator recanalization rates, suggesting an impairment of the fibrinolytic system by hyperglycaemia.

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A few studies investigated the relation between venous thromboembolism (VTE) and hyperglycaemia. Pre-surgery hyperglycaemia (>11.1 mmol/l) was associated with an OR of 3.2 for pulmonary embolism after orthopaedic surgery65_{. However, this might reflect}

undiagnosed and uncontrolled diabetes. Recently, we published data on glucose levels at presentation for suspected VTE and showed that higher glucose levels at presentation are associated with actually having VTE, in a clear dose-response fashion66_{. During hip}

surgery, glucose levels rise in patients without diabetes and this precedes a rise in factor VIII, vWF and F1+267_{, but further investigation is needed to confirm that this is a causal}

relationship.

Whether intensive treatment of acute hyperglycaemia improves clinical outcome in these thrombotic conditions remains unknown. Although the DIGAMI trial showed a beneficial clinical effect of intensive blood glucose control by glucose-insulin-potassium (GIK) infusion in MI patients with diabetes, these results could not be reproduced in patients without diabetes due to a lack of contrast in blood glucose values between intervention and control groups68-71_{. The GIST-UK trial randomized stroke patients to}

GIK infusion or saline infusion to investigate the effect of glucose modulation by GIK. In this trial no clinical benefit was observed either, but the trial was underpowered, the glucose lowering effect of GIK was small and patients were treated for only 24 hours72_.

Clinical trials investigating the effect of glucose control on VTE development are yet to be performed.

Possible mechanisms

Many theories on how hyperglycaemia leads to hypercoagulability have already been proposed and studied73_{. First, on a cellular level, hyperglycaemia and also}

hyperinsulinaemia increases the expression of PAI-1 on vascular smooth muscle cells

in vitro, thereby increasing its concentration and activity. As a result, the activity of t-PA

is reduced thereby decreasing the fibrinolytic potential74_{. The authors suggested that a}

direct effect of glucose and insulin on gene transcription could be responsible. Indeed, hyperglycaemia in the presence of insulin increases the activity of transcription factor nuclear factor kappa-B (NF-kB) in human hepatocyte cells and the gene transcription of PAI-1 in vitro, which suggests that PAI-1 transcription is increased via NF-kB75_{. Because}

this effect disappeared when an antioxidant was added to the medium, hyperglycaemia-induced oxidative stress was hypothesized to be the major activator of NF-kB. In line, Khechai et al.76_{studied the effect of advanced glycation end products (AGE) on}

TF expression in human monocytes and concluded AGE induced TF expression at the mRNA level, which could be diminished by adding antioxidants. The effect of AGEs on coagulation activation was also seen when human umbilical vein endothelial cells were exposed to AGEs77_{and AGEs dose-dependently increased procoagulant activity and TF}

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levels. In vivo, withholding insulin in patients with DM1 increased TF and PAI-1 levels, which was accompanied by a rise in malondialdehyde (MDA) and protein carbonyl groups (PCG), both markers of oxidative stress25_.

Next, hyperglycaemia directly influences the vulnerability of the vascular endothelium by affecting the glycocalyx, a protective layer of proteoglycans covering the vessel wall. This results in enhanced platelet-endothelial cell adhesion and release of coagulation factors harboured within the endothelial glycocalyx78_{. What is more, hyperglycaemia}

is known to cause increased glycation of proteins and this may also occur in proteins involved in coagulation and fibrinolysis. Verkleij et al. 7_{showed that glycated TAFI}

loses its fibrinolytic properties in vitro, although this could not be reproduced in vivo. Fibrin clots from patients with diabetes type 2 are denser as compared with controls and displayed an altered structure, resulting in longer clot lysis time79_{. In vivo glycaemic}

control was directly correlated to the clot density from the patients. A likely explanation for this is the possible non-enzymatic glycation of fibrin80_{. It is conceivable that other}

coagulation proteins are also glycated, altering their activity.

It is not entirely clear how hyperinsulinaemia adds to the prothrombotic effects of hyperglycaemia. However, several studies have shown an independent effect of both hyperinsulinaemia and hyperglycaemia on thrombotic markers and an additive effect when simultaneously present, as in DM2 or acute hyperglycaemia14, 44, 74_{. What is more,}

the insulin resistant state that underlies these metabolic conditions has been described to extend to blood platelet activity as well. Whereas insulin has an inhibiting effect on platelet activation and aggregation in healthy individuals, there are several studies that show platelets of DM2 patients to be resistant to this inhibitory effect of insulin, making them more susceptible to activation81-83_.

Thus, multiple complex pathways are likely to be involved in the induction of hypercoagulability by hyperglycaemia, and its effect is more profound in combination with hyperinsulinaemia. Although we separately discussed the effects of chronic and acute hyperglycaemia on coagulation and fibrinolysis, the current evidence gives no reason to assume that the underlying mechanisms differ. Nevertheless, the models used to study the effects of chronic and acute hyperglycaemia are quite different and not easily comparable.

Summary

In summary, laboratory evidence is suggestive of a contribution of both chronic and acute hyperglycaemia to coagulation activation and hypofibrinolysis, resulting in a procoagulant state (Figure 1). What is more, hyperglycaemia is often accompanied by hyperinsulinaemia and their combined effects may result in an even stronger hypercoagulable state.

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Evidence for the clinical consequences of these prothrombotic alterations during hyperglycaemia is still circumstantial. However, intensive glycaemic control in patients with diabetes reduced the incidence of thrombotic diseases such as myocardial infarction and stroke in the long run. Whether intensive glucose control during acute hyperglycaemia, acute MI, stroke and VTE could prevent hypercoagulability and thereby improve outcome awaits further investigation in randomized controlled trials.

Figure 1. A simplified impression of the relationship between hyperglycaemia, hyperinsulinaemia and

coagulation, leading to clinical outcome.

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