In the Journals

Anticoagulant effect of aspirin goes beyond platelet aggregation

Aspirin’s many mechanisms of action against cardiovascular events may also help to explain reported resistance to therapy.

Aspirin’s effect on homeostasis is well-known. Low-dose aspirin (acetylsalicylic acid, 81 mg) inhibits the enzyme Cox-1, which produces thromboxane A-2, necessary for platelet aggregation.

“The primary effect of aspirin as an anticoagulant is thought to involve platelet function; however, aspirin is also an anti-inflammatory,” said Kenneth Mann, PhD, a professor from the department of biochemistry at the University of Vermont.

Less clear are other methods by which aspirin acts as an anticoagulant. In a review article published in Blood, Mann, Anetta Undas, MD, PhD, and colleagues presented an overview of other possible antithrombotic properties of aspirin.

Thrombin formation

Thrombin (activated Factor II [IIa]), a serine protease, converts fibrinogen into insoluble strands of fibrin. Fibrin, a protein, crosslinks with Factor XIII enzyme (fibrin stabilizing Factor FXIII) and combines with platelets to form a clot.

Studies of microvascular injury models have demonstrated that aspirin at a daily dose of 30 mg administered for one week decreased thrombin formation in healthy patients. Aspirin at higher doses (75 mg and 300 mg) decreased concentrations of thrombin markers similarly, as did a single dose of 500 mg following a period of aspirin therapy. This thrombin-lowering effect was found in healthy individuals and patients with increased risk for coronary artery disease.

A seven-day course of low-dose (75 mg) aspirin was associated with slower prothrombin consumption (by 29%), thrombin formation (by 27.2%) and prothrombinase formation (by 29%) at the site of microvascular injury.

Aspirin also slowed FXIII activation by thrombin and decreased the maximum rate of FXIII cleavage. The researchers pointed out that studies on the direct effects of high-dose aspirin on plasma and blood coagulation in vitro have provided mixed results.

Inhibition of tissue factor

The exposure of exposed/expressed tissue factor in the subendothelium triggers in vivo coagulation. According to the researchers, studies suggest that aspirin may inhibit exposed/expressed tissue factor synthesis in monocytes and reduce tissue factor expression in artheosclerotic plaques.

“The inflammatory and coagulation processes are linked; for example, the protein tissue factor is associated both with the inflammatory response and coagulation, and tissue factor is the primary initiator of the coagulation system,” Mann said in an interview.

“The anti-inflammatory effect of chronic aspirin administration also probably down-regulates tissue factor presentation by inflammatory cells in the blood circulation. It also potentially alters tissue factor presentation by the vascular endothelial cells. The inflammation-coagulation processes are areas of active investigation. Hopefully the relationships will be resolved by improving technologies over the next few years,” he said.

Polymorphisms and fibrin

When aspirin inhibits thrombin generation, it subsequently inhibits the creation of fibrin on arterial walls, thus interrupting hemostasis. Upon introduction into fibrin, aspirin may interfere with FXIII activation and function because fibrinogen and fibrin enhance FXIII activation approximately 100-fold. Genetic polymorphisms influenced by aspirin may consequently alter the stability of the fibrin network.

The Val34Leu polymorphism in the A chain of FXIII is in close proximity to the thrombin cleavage site at Arg37-Glyl38. It has been suggested that this mutation may influence FXIII activation due to its relative position. The researchers showed that a seven-day administration of 75 mg aspirin per day inhibits FXIII activation to a greater degree in LEU34–positive healthy patients compared with patients with the Val34Val genotype.

Mann and colleagues cited a study of fibrin clot properties in healthy individuals with three allelic variants of the FXIII Val34Leu mutation before and after they received 300 mg aspirin. After four hours, patients with the LEU34 allele exhibited significantly greater clot permeability, although permeability increased in association with the Val34Leu polymorphism to some degree in all patients.

The researchers suggested that aspirin alters fibrin cross-linking to a greater degree in individuals with the LEU34 allele compared with individuals with the Val34Val genotype. Therefore, a pharmacogenetic association might exist between aspirin’s antithrombotic effects and the presence of the FXIII LEU34 allele, according to the researchers.

A common polymorphism of the B3-integrin gene, PIA1A2, may modulate the effect of aspirin-related alterations in thrombin formation. Studies have found impaired platelet aggregation in patients with the PI*A2 allele who were treated with aspirin.

Other methods of modulation

In vitro and in vivo studies have demonstrated that fibrinogen is acetylated with administration of high dose aspirin (650 mg every 12 hours). In vivo, acetylation of fibrinogen alters its structure, thus altering properties of the subsequent blood clot. In individuals taking 650 mg aspirin twice daily, the extent of acetylation in fibrinogen inversely correlated with clot lysis time. Other studies have shown that aspirin changes fibrin gel porosity and affects clot permeability.

Aspirin and cholesterol

The researchers noted a positive correlation between total cholesterol or low-density lipoprotein cholesterol and the amount of thrombin generated after aspirin administration. Studies showed that 75 mg aspirin daily reduced thrombin formation only in patients whose total cholesterol level was below 200 mg/dL. In patients with total cholesterol levels between 200 mg/dL and 250 mg/dL, low-dose aspirin did not appear to impair thrombin formation. However, 300 mg aspirin daily has shown to inhibit thrombin generation in patients with a total cholesterol less than 240 mg/dL and LDL cholesterol less than 155 mg/dL.

Researchers suggested that there is enough evidence to conclude that aspirin affects blood coagulation on many levels, not simply platelet function. Aspirin may reduce thrombin formation and subsequently affect fibrin production. It may also affect acetylate fibrinogen, resulting in increased clot permeability and lysis, according to the researchers.

“The qualitative and quantitative properties of the blood pro- and anti-coagulation proteins are highly variable within the population because of both genetic and environmental factors. Platelet function is also highly variable,” said Mann. “It is not surprising that differential protective effects of aspirin are observed over the entire population.”

The researchers concluded that these additional anticoagulant effects of aspirin are increasingly important as aspirin becomes more widely used in antithrombotic therapy. – by Carey Cowles

Editor’s note: These important findings provide insights into previous findings that anticoagulant effects of aspirin in baboons are markedly amplified if multiple administrations per day are provided. This was inexplicable if only aspirin’s irreversible effect on platelet function is considered. Moreover, the reviewed data in this article suggests that higher doses of aspirin than present in ‘baby aspirins, provided several times daily, should now be considered for atherosclerosis prophylaxis. – Harry S. Jacob, MD
For more information:
  • Undas A, Brummel-Ziedins K, Mann K. Antithrombotic properties of aspirin and resistance to aspirin: beyond strictly antiplatelet actions. Blood. 2007;109:2285-2292.

Aspirin’s effect on homeostasis is well-known. Low-dose aspirin (acetylsalicylic acid, 81 mg) inhibits the enzyme Cox-1, which produces thromboxane A-2, necessary for platelet aggregation.

“The primary effect of aspirin as an anticoagulant is thought to involve platelet function; however, aspirin is also an anti-inflammatory,” said Kenneth Mann, PhD, a professor from the department of biochemistry at the University of Vermont.

Less clear are other methods by which aspirin acts as an anticoagulant. In a review article published in Blood, Mann, Anetta Undas, MD, PhD, and colleagues presented an overview of other possible antithrombotic properties of aspirin.

Thrombin formation

Thrombin (activated Factor II [IIa]), a serine protease, converts fibrinogen into insoluble strands of fibrin. Fibrin, a protein, crosslinks with Factor XIII enzyme (fibrin stabilizing Factor FXIII) and combines with platelets to form a clot.

Studies of microvascular injury models have demonstrated that aspirin at a daily dose of 30 mg administered for one week decreased thrombin formation in healthy patients. Aspirin at higher doses (75 mg and 300 mg) decreased concentrations of thrombin markers similarly, as did a single dose of 500 mg following a period of aspirin therapy. This thrombin-lowering effect was found in healthy individuals and patients with increased risk for coronary artery disease.

A seven-day course of low-dose (75 mg) aspirin was associated with slower prothrombin consumption (by 29%), thrombin formation (by 27.2%) and prothrombinase formation (by 29%) at the site of microvascular injury.

Aspirin also slowed FXIII activation by thrombin and decreased the maximum rate of FXIII cleavage. The researchers pointed out that studies on the direct effects of high-dose aspirin on plasma and blood coagulation in vitro have provided mixed results.

Inhibition of tissue factor

The exposure of exposed/expressed tissue factor in the subendothelium triggers in vivo coagulation. According to the researchers, studies suggest that aspirin may inhibit exposed/expressed tissue factor synthesis in monocytes and reduce tissue factor expression in artheosclerotic plaques.

“The inflammatory and coagulation processes are linked; for example, the protein tissue factor is associated both with the inflammatory response and coagulation, and tissue factor is the primary initiator of the coagulation system,” Mann said in an interview.

“The anti-inflammatory effect of chronic aspirin administration also probably down-regulates tissue factor presentation by inflammatory cells in the blood circulation. It also potentially alters tissue factor presentation by the vascular endothelial cells. The inflammation-coagulation processes are areas of active investigation. Hopefully the relationships will be resolved by improving technologies over the next few years,” he said.

Polymorphisms and fibrin

When aspirin inhibits thrombin generation, it subsequently inhibits the creation of fibrin on arterial walls, thus interrupting hemostasis. Upon introduction into fibrin, aspirin may interfere with FXIII activation and function because fibrinogen and fibrin enhance FXIII activation approximately 100-fold. Genetic polymorphisms influenced by aspirin may consequently alter the stability of the fibrin network.

The Val34Leu polymorphism in the A chain of FXIII is in close proximity to the thrombin cleavage site at Arg37-Glyl38. It has been suggested that this mutation may influence FXIII activation due to its relative position. The researchers showed that a seven-day administration of 75 mg aspirin per day inhibits FXIII activation to a greater degree in LEU34–positive healthy patients compared with patients with the Val34Val genotype.

Mann and colleagues cited a study of fibrin clot properties in healthy individuals with three allelic variants of the FXIII Val34Leu mutation before and after they received 300 mg aspirin. After four hours, patients with the LEU34 allele exhibited significantly greater clot permeability, although permeability increased in association with the Val34Leu polymorphism to some degree in all patients.

The researchers suggested that aspirin alters fibrin cross-linking to a greater degree in individuals with the LEU34 allele compared with individuals with the Val34Val genotype. Therefore, a pharmacogenetic association might exist between aspirin’s antithrombotic effects and the presence of the FXIII LEU34 allele, according to the researchers.

A common polymorphism of the B3-integrin gene, PIA1A2, may modulate the effect of aspirin-related alterations in thrombin formation. Studies have found impaired platelet aggregation in patients with the PI*A2 allele who were treated with aspirin.

Other methods of modulation

In vitro and in vivo studies have demonstrated that fibrinogen is acetylated with administration of high dose aspirin (650 mg every 12 hours). In vivo, acetylation of fibrinogen alters its structure, thus altering properties of the subsequent blood clot. In individuals taking 650 mg aspirin twice daily, the extent of acetylation in fibrinogen inversely correlated with clot lysis time. Other studies have shown that aspirin changes fibrin gel porosity and affects clot permeability.

Aspirin and cholesterol

The researchers noted a positive correlation between total cholesterol or low-density lipoprotein cholesterol and the amount of thrombin generated after aspirin administration. Studies showed that 75 mg aspirin daily reduced thrombin formation only in patients whose total cholesterol level was below 200 mg/dL. In patients with total cholesterol levels between 200 mg/dL and 250 mg/dL, low-dose aspirin did not appear to impair thrombin formation. However, 300 mg aspirin daily has shown to inhibit thrombin generation in patients with a total cholesterol less than 240 mg/dL and LDL cholesterol less than 155 mg/dL.

Researchers suggested that there is enough evidence to conclude that aspirin affects blood coagulation on many levels, not simply platelet function. Aspirin may reduce thrombin formation and subsequently affect fibrin production. It may also affect acetylate fibrinogen, resulting in increased clot permeability and lysis, according to the researchers.

“The qualitative and quantitative properties of the blood pro- and anti-coagulation proteins are highly variable within the population because of both genetic and environmental factors. Platelet function is also highly variable,” said Mann. “It is not surprising that differential protective effects of aspirin are observed over the entire population.”

The researchers concluded that these additional anticoagulant effects of aspirin are increasingly important as aspirin becomes more widely used in antithrombotic therapy. – by Carey Cowles

Editor’s note: These important findings provide insights into previous findings that anticoagulant effects of aspirin in baboons are markedly amplified if multiple administrations per day are provided. This was inexplicable if only aspirin’s irreversible effect on platelet function is considered. Moreover, the reviewed data in this article suggests that higher doses of aspirin than present in ‘baby aspirins, provided several times daily, should now be considered for atherosclerosis prophylaxis. – Harry S. Jacob, MD
For more information:
  • Undas A, Brummel-Ziedins K, Mann K. Antithrombotic properties of aspirin and resistance to aspirin: beyond strictly antiplatelet actions. Blood. 2007;109:2285-2292.