Thrombotic abnormalities in diabetes

Diabetes is strongly linked with cardiovascular disease (CVD). In addition to increased risk of myocardial infarction, the prognosis following a vascular event in diabetes remains worse than in individuals with normal glucose metabolism. This association is related to clustering of cardiovascular risk factors in diabetes, coupled with a deranged haemostatic system that is driven by inflammation, insulin resistance and hyperglycaemia. A pro-thrombotic environment is generated secondary to a number of abnormalities including increased platelet activation and elevated levels of proteins such as coagulation factors VII and VIII, fibrinogen and plasminogen activator inhibitor 1 (PAI-1). Many of these changes lead to an alteration in the fibrin network, the backbone of the obstructive vascular thrombus, resulting in a denser structure with thinner fibrin fibres which further contribute to hypofibrinolysis.

Background

The formation of blood clots and their subsequent destruction, or lysis, (termed haemostasis) is a vital physiological function and a fine balance of interactions between the vessel wall (in particular the endothelium) and platelets (cellular phase), along with procoagulant and anticoagulant proteins (fluid phase). The Figure 1 [Click to enlarge]. Simplified illustration of fibrin clot formation and fibrinolysis.  Following plaque rupture or fissure, a TF/FVII complex is formed and platelets are activated. This results in the activation of numerous coagulation factors and ultimately the conversion of prothrombin to thrombin, which converts soluble fibrinogen to fibrin. A network of fibrin fibres is formed which is stabilised by the actions of FXIII, cross linking the fibres. Clot lysis occurs following the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) which generates fibrin degradation products. Fibrinolysis is regulated by plasminogen activator inhibitor-1(PAI-1) which inhibits tPA and α2-antiplasmin (α2-ap) which inhibits plasmin activity.
Figure 1 [Click to enlarge]. Simplified illustration of fibrin clot formation and fibrinolysis. Following plaque rupture or fissure, a TF/FVII complex is formed and platelets are activated. This results in the activation of numerous coagulation factors and ultimately the conversion of prothrombin to thrombin, which converts soluble fibrinogen to fibrin. A network of fibrin fibres is formed which is stabilised by the actions of FXIII, cross linking the fibres. Clot lysis occurs following the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) which generates fibrin degradation products. Fibrinolysis is regulated by plasminogen activator inhibitor-1(PAI-1) which inhibits tPA and α2-antiplasmin (α2-ap) which inhibits plasmin activity.
resultant plug of platelets, red blood cells, leucocytes and fibrin fibres seals the vessel preventing leakage of blood from the vascular tree, and permits wound healing.

In contrast to this, thrombosis is a pathological state, where blood clot formation in a diseased blood vessel results in partial or complete blood flow obstruction, resulting in end organ damage. Clot formation is initiated by tissue factor (TF) on the surface of damaged endothelial cells. A cascade of enzymatic reactions ensues, that activate a number of coagulation factors (F), ultimately resulting in the generation of thrombin, which has two important functions, firstly converting soluble fibrinogen to insoluble fibrin and secondly activating FXIII, which stabilises the fibrin clot through cross linking of fibrin fibres.

The process is regulated by the fibrinolytic pathway, which is activated upon fibrin formation, and helps localise clot formation and initiates clot lysis through the actions of plasmin (Figure 1).

Chronic inflammation associated with diabetes results in endothelial cell dysfunction and ultimately the development of atherosclerotic plaques that have an increased tendency to rupture which triggers the sequence of events as described above, forming a platelet rich, fibrin mesh. This scenario often resolves with no clinical sequelae; however vessel occlusion frequently occurs leading to clinical entities such as acute myocardial infarction, unstable angina or stroke. Not only is diabetes associated with increased plaque formation and vulnerability to rupture: a pro-thrombotic environment exists which underlies the increased risk of cardiovascular disease.

Effect of diabetes on haemostasis

The mechanisms involved in creating the pro-thrombotic environment associated with diabetes are multiple. Insulin resistance and inflammation play a key role, leading to increased production of several coagulation proteins and also anti-fibrinolytic molecules, along with increased platelet activation.

Hyperglycaemia itself also contributes to these abnormalities and complicates matters further by glycation of proteins such as fibrinogen which unfavourably alters the structure of the fibrin clot. The effects of diabetes on various compounds involved in haemostasis will be discussed below and are summarised in Table 1.

Tissue Factor

Tissue factor (TF) is responsible for the initial activation of coagulation. Diabetes is associated with elevated levels of TF, possibly as a result of diminished inhibition of TF synthesis by insulin in diabetes compared with non-diabetes subjects [1].

Factor VII

Factor VII forms a complex with TF and initiates the formation of thrombin. Levels are raised in type 2 diabetes and are known to correlate with plasma triglyceride levels. Possible explanations for elevated FVII levels may relate to increased triglyceride levels associated with diabetes as FVII binds to very low density lipoprotein (VLDL) particles rich in triglycerides and reduced post-prandial breakdown of these particles results in increased plasma FVII levels[2]. Dyslipidaemia associated with diabetes may also increase plasma FVII through increased production of kallikrein.

von Willebrand factor and Factor VIII

von Willebrand factor (vWf), secreted by vascular endothelial cells, facilitates platelet adhesion following disruption to endothelial cell integrity and exposure of the sub-endothelium. It is bound in plasma to clotting factor VIII and elevated levels are seen in diabetes. These changes are likely to represent endothelial cell damage, inflammation and insulin resistance.[3]

Thrombin

Activated thrombin plays a crucial role in the conversion of soluble fibrinogen to insoluble fibrin and ultimately a cross linked fibrin clot structure. Hyperglycaemia is linked with increased thrombin production and elevated levels are seen in type 1 and type 2 diabetes which can be lowered by improved glycaemic control[4]. Together with increased clot formation, elevated thrombin levels contribute to the formation of a denser fibrin clot that is more resistant to fibrinolysis.

Fibrinogen

Figure 2. Electron Microscopy of ex-vivo fibrin clots. Top image healthy control, bottom image diabetes patient. The diabetes clot is denser with thinner fibrin fibres and fewer pores between the fibres. These clots are increasingly resistant to fibrinolysis compared to non-diabetes clots and may reflect reduced plasmin generation due to increased incorporation of α2-ap.
Figure 2. Electron Microscopy of ex-vivo fibrin clots. Top image healthy control, bottom image diabetes patient. The diabetes clot is denser with thinner fibrin fibres and fewer pores between the fibres. These clots are increasingly resistant to fibrinolysis compared to non-diabetes clots and may reflect reduced plasmin generation due to increased incorporation of α2-ap.
Fibrinogen is synthesised in the liver and following activation of the coagulation pathway is converted to a fibrin monomer (by thrombin) which subsequently attaches to neighbouring molecules through lateral aggregation, forming the backbone of the blood clot. Elevated levels of fibrinogen are observed in diabetes and likely reflects increased hepatic production through the actions of inflammatory cytokines and insulin[5]. Plasma levels of fibrinogen affect thrombus formation and elevated levels are known to be a strong and independent risk factor for the development of CVD. In vivo hyperglycaemia in poorly controlled diabetes patients is known to cause glycation of fibrinogen[6] which causes an alteration in the structure and function of the molecule and contributes to the ex-vivo formation of a rigid and dense clot with thinner fibrin fibres and fewer pores (Figure 2). These clots are increasingly resistant to fibrinolysis compared to non-diabetes clots and are associated with increased cardiovascular risk.

FXIII

In diabetes there is increased cross linking of an important anti-fibrinolytic enzyme, α2-antiplasmin (α2-ap), to fibrinogen during clot formation[7]. The mechanism underpinning this has not been elucidated but it may be related to structural changes that occur within the fibrinogen molecule as a result of hyperglycaemia or through increased activation of FXIII by thrombin.

Plasminogen and Plasmin

Plasminogen is converted to plasmin, a key fibrinolytic enzyme, by the actions of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Plasmin is responsible for the dissolution of fibrin fibres within the clot, generating fibrin degradation products. Increased glycation of plasminogen has been observed in diabetes compared with healthy controls, which is thought to reduce conversion to plasmin, along with the reducing the efficacy of plasmin itself once generated[8], contributing to the hypofibrinolytic environment associated with diabetes.

Plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) serves to regulate fibrinolysis by interfering with the conversion of plasminogen to plasmin. It irreversibly binds to, and inhibits tPA and uPA, thereby reducing plasmin generation and modifying the rate of clot lysis. In diabetes subjects there are increased levels of PAI-1, which is positively correlated with glycaemic control[9]9, and elevated levels are also thought to be an independent risk factor for the development of type 2 diabetes. A combination of inflammatory cytokines such as tumour necrosis factors alpha (TNFα), insulin, free fatty acids, glucose and VLDL are likely to cause increased release of PAI-1 from the liver and adipocytes.

Effect of diabetes on platelets

Platelets play a crucial role in the formation of the fibrin clot, from the conversion of prothrombin to thrombin, through to their interaction with fibrin fibres and aggregation via glycoprotein receptors (GPIIb/IIIa). Adhesion of the platelet to the endothelial wall allows activation and the release a variety of compounds from storage granules that facilitate further platelet adhesion and activation, promote tissue repair and initiate platelet aggregation.

These actions are closely regulated by a number of molecules, and in the context of diabetes the most important is Nitric Oxide (NO). Under normal conditions, NO inhibits platelet adherence to the endothelial wall, thus preventing the generation of thrombi in healthy vessels, as well as promoting vasodilation. The bioavailability of NO is reduced in type 2 diabetes and is largely a reflection of underlying endothelial cell damage and increased reactive oxygen species associated with hyperglycaemia. This results in increased platelet activation and consequently increased thrombus formation and vasoconstriction[2]

Table 1. Summary of changes to haemostatic components in diabetes

Haemostatic component Function Changes in DM Effect in DM
TF Initiates clotting cascade ↑levels ↑ thrombosis
FVII Forms complex with TF ↑levels ↑thrombosis
FVIII & vWf complex Adherence of platelets to endothelial cell wall ↑levels ↑platelet activation
Thrombin Converts fibrinogen to fibrin ↑ levels Altered clot structure
Fibrinogen Forms fibrin clot ↑levels / ↑glycation Altered clot structure/↓fibrinolysis
Plasmin Breaks down fibrin clot ↓levels ↓fibrinolysis
PAI-1 Inhibits production of plasmin ↑levels ↓fibrinolysis
Platelets Activation of coagulation factors and forms fibrin mesh ↑activation ↑thrombosis

Conclusion

As detailed above, diabetes is associated with various prothrombotic abnormalities, yet there are no diabetes-specific therapies to reduce thrombosis risk in this condition. One difficulty is the heterogeneous nature of diabetes with varying degrees of thrombosis potential, necessitating tailored therapy for optimal effect. Future research is needed to fully understand the mechanistic pathways implicated in thrombosis risk in diabetes, with special emphasis on identifying groups in whom intensification of anti-thrombotic therapy is beneficial. Moreover, given the major changes observed within the fluid phase of coagulation in diabetes, future therapies should explore targeting this arm of coagulation rather than concentrating on anti-platelet therapy alone.

References

  1. ^ Gerrits AJ et al. Platelet tissue factor synthesis in type 2 diabetic patients is resistant to inhibition by insulin. Diabetes 2010;59:1487-1495.

  2. ^ Grant PJ. Diabetes mellitus as a prothrombotic condition. J.Intern.Med. 2007;262:157-172.

  3. ^ Pinkney JH et al. Endothelial dysfunction: cause of the insulin resistance syndrome. Diabetes 1997;46 Suppl 2:S9-13.

  4. ^ Undas A et al. Hyperglycemia is associated with enhanced thrombin formation, platelet activation, and fibrin clot resistance to lysis in patients with acute coronary syndrome. Diabetes Care 2008;31:1590-1595.

  5. ^ Ajjan R, Grant PJ. Coagulation and atherothrombotic disease. Atherosclerosis 2006;186:240-259.

  6. ^ Pieters M, van Zyl DG, Rheeder P et al. Glycation of fibrinogen in uncontrolled diabetic patients and the effects of glycaemic control on fibrinogen glycation. Thromb.Res. 2007;120:439-446.

  7. ^ Agren A, Jorneskog G, Elgue G et al. Increased incorporation of antiplasmin into the fibrin network in patients with type 1 diabetes. Diabetes Care 2014;37:2007-2014.

  8. ^ Ajjan RA, Gamlen T, Standeven KF et al. Diabetes is associated with post-translational modifications in plasminogen resulting in reduced plasmin generation and enzyme specific activity. Blood 2013

  9. ^ Seljeflot I et al. Fibrinolytic activity is highly influenced by long-term glycemic control in Type 1 diabetic patients. J Thromb Haemost 2006;4:686-688.

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