Comment on Dayal et al, page 2237

Dayal and colleagues demonstrate enhanced susceptibility to arterial occlusion in a murine model of hyperhomocysteinemia in the absence of evidence of altered platelet function. These investigators provide evidence that ROS generation and/or a decrease in localized activation of the protein C anticoagulation pathway may contribute to enhanced thrombosis in this model.

Hyperhomocysteinemia has long been recognized as a cardiovascular risk factor with continuing controversy regarding whether it is a marker of or a cause of disease.1,2  Metabolism of homocysteine relies on folate and vitamin B-dependent enzymes, including cystathionine β-synthase (CBS). To produce hyperhomocysteinemia in mice, Dayal and colleagues use both genetic (Cbs heterozygous deficient mice [Cbs+/–]) and dietary means (high methionine/low folate [HM/LF] diets). Using the photochemical injury model to promote thrombosis, these investigators found that diet had a more profound effect than genotype in enhancing arterial occlusion. One explanation is a threshold effect, as HM/LF diet groups (Cbs+/+ or Cbs+/–) have the highest levels of homocysteine. Alternatively, low folate or high methionine could contribute to enhanced vascular occlusion in this model. The important question is, how does elevated homocysteine enhance thrombosis? Possibilities include increased generation of reactive oxygen species (ROS) followed by subsequent loss of bioavailable nitric oxide (NO), NO being a naturally occurring inhibitor of platelet adhesion and activation. Other possibilities include platelet activation, alterations in prothrombotic factors such as tissue factor, or decreased expression/activation of antithrombotic factors such as tissue plasminogen activator and protein C.

Dayal and colleagues investigated many of these mechanisms. As expected, hyperhomocysteinemia leads to an increase in vascular ROS. To determine a source for increased ROS production, Dayal and colleagues investigated whether the expression of NADPH oxidase subunits was altered with hyperhomocysteinemia. They found an increase in expression of the NOX4 subunit in vascular tissue, which may lead to increased superoxide production; however, the mechanism by which reactive oxygen species enhance thrombosis is unresolved. These investigators down-play the importance of reduced bioavailable NO in promoting thrombosis on the basis of indirect evidence: thrombosis in endothelial NO synthase (eNOS)–deficient mice. In agreement with recently published work, lack of eNOS did not accelerate vascular occlusion. Previously, Iafrati et al3  characterized compensatory up-regulation of antithrombotic factors in eNOS-deficient mice that may, in part, explain these unexpected findings. Additional studies are necessary to clarify the thrombotic effects of NO deficiency in the context of hyperhomocysteinemia and to address the contribution of ROSs to thrombotic susceptibility in this model. In the current report, 2 other oxidant-sensitive prothrombotic changes, platelet activation and tissue factor expression, were not significantly altered by hyperhomocysteinemia.

In contrast to the in vitro platelet studies by Dayal and colleagues, Weiss et al4  showed that mildly hyperhomocysteinemic mice have higher circulating levels of P-selectin, indicating that platelets are activated in vivo by elevated homocysteine. Importantly, Dayal and colleagues found that washed platelets, free from the hyperhomocysteinemic environment, are not altered in their response to thrombin activation. These data implicate nonplatelet factors for enhanced coagulation in this model. One potential candidate is the anticoagulant protein C.

To analyze the protein C anticoagulation pathway, Dayal and colleagues investigated the activation of protein C as well as vascular expression of factors that are involved in activating protein C, endothelial protein C receptor (EPCR) and thrombomodulin. Although systemic activation of protein C was unchanged, Dayal and colleagues found that hyperhomocysteinemia decreases activation of protein C in vascular tissue, whereas transcripts for EPCR and thrombomodulin are increased. The protein C story is complex, as coagulation is affected not only by levels of activated protein C but also by its inhibitors. Known inhibitors include a soluble form of EPCR,5  which is shed from the membrane in response to thrombin and inflammatory mediators. The studies by Dayal and colleagues implicate ROS and protein C, 2 known mediators of coagulation and thrombosis; however, further analysis of these and other factors is necessary to resolve the prothrombotic actions of homocysteine. ▪

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