Intravenous infusion of recombinant tPA is a well-established thrombolytic therapy used in the treatment of thrombotic cardiovascular diseases, such as ischemic stroke and myocardial infarction.
Recent research has shown that low plasma tPA activity is associated with an increased risk of atherosclerotic cardiovascular diseases and elevated levels of atherogenic apolipoprotein B (apoB)-lipoprotein cholesterol.
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Tissue plasminogen activator (tPA) is a protein that helps to break down blood clots. It is produced by the liver and released into the bloodstream when there is a clot. Low plasma tPA activity means that there is less tPA available to break down blood clots. This can increase the risk of atherosclerotic cardiovascular diseases, such as heart attack and stroke.
Atherosclerosis is a condition in which plaque builds up in the walls of the arteries. This plaque can narrow the arteries and make it difficult for blood to flow through them. If a blood clot forms in a narrowed artery, it can block the flow of blood and cause a heart attack or stroke.
Apolipoprotein B (apoB) is a protein that is found in low-density lipoprotein (LDL) cholesterol, also known as “bad” cholesterol. LDL cholesterol can build up in the walls of the arteries and contribute to the development of atherosclerosis.
Studies have shown that people with low plasma tPA activity are more likely to have high levels of apoB-lipoprotein cholesterol. This suggests that low plasma tPA activity may be a risk factor for atherosclerosis.
The exact mechanism by which low plasma tPA activity increases the risk of atherosclerosis is not fully understood. However, it is thought that low tPA activity may make it more difficult to break down blood clots that form in the arteries. This can lead to the development of plaque and narrowing of the arteries.
However, the precise relationship between low tPA levels and apoB-lipoprotein cholesterol remains unclear.
Results: To explore the role of tPA in apoB-lipoprotein metabolism, experiments were conducted in mice where tPA expression was either silenced or deleted in hepatocytes. These genetic manipulations resulted in higher levels of plasma apoB and cholesterol, independent of any changes in hepatic low-density lipoprotein receptor (LDLR) or apolipoprotein E (apoE) expression or the mRNA levels of Apob.
Notably, the elevated cholesterol in these mice was distributed within the very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions. Similarly, silencing tPA in human primary hepatocytes led to increased secretion of newly synthesized apoB, despite no alterations in Apob mRNA levels. These findings collectively suggest that a deficiency in hepatocyte-derived tPA promotes the secretion of apoB-lipoproteins.
The lipidation status of apoB is a crucial determinant of its intracellular fate. Poorly lipidated apoB is subject to intracellular degradation, whereas fully lipidated apoB is efficiently secreted as larger-sized, lower-density VLDL particles. Silencing hepatocyte tPA in mice lacking LDL receptors (Ldlr−/−) led to the production of larger VLDL particles in the bloodstream, containing a higher triglyceride content, indicating enhanced intrahepatic apoB lipidation.
In hepatocytes, apoB lipidation occurs in the endoplasmic reticulum (ER) through the action of microsomal triglyceride transfer protein (MTP), which incorporates neutral lipids into nascent apoB. Intriguingly, when human primary hepatocytes were transfected with a plasmid encoding tPA, there was a reduction in the secretion of newly synthesized apoB labeled with [3H].
Further investigations using proximity ligation, confocal imaging, and immunoprecipitation assays revealed that tPA physically interacts with apoB within the hepatocyte ER. Moreover, recombinant tPA was found to interact with LDL immobilized on solid surfaces, inhibiting the interaction between apoB and MTP and reducing the transfer of neutral lipids to apoB.
Importantly, even the serine protease inactive form of tPA (S513A) exhibited the same inhibitory effects on apoB secretion and lipid transfer, indicating that this action of tPA is independent of its protease activity. The interaction between tPA and LDL was blocked by antibodies targeting the Kringle 2 (K2) domain of tPA, the MTP-interacting regions at the N terminus of apoB, and the lysine analog tranexamic acid.
Additionally, deletion of the K2 domain or mutation of the lysine-binding site within the K2 domain of tPA abolished the inhibitory effects of tPA on apoB secretion. These findings collectively demonstrate that tPA, through its K2 domain’s lysine-binding site, binds to apoB’s N terminus, interfering with the apoB-MTP interaction within hepatocytes. This disruption ultimately results in reduced VLDL assembly and lower plasma levels of atherogenic apoB-lipoprotein cholesterol.
Under conditions of lipid loading in hepatocytes, PAI-1 forms a complex with tPA, sequestering tPA away from apoB. This sequestration allows apoB to undergo lipidation efficiently and facilitates VLDL assembly and secretion. Human subjects with PAI-1 deficiency exhibit smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol, further supporting the importance of the tPA-PAI-1 interaction in hepatic lipoprotein regulation.
Conclusion: This study sheds light on a finely-tuned mechanism of VLDL assembly within hepatocytes, which involves intricate interactions among tPA, PAI-1, and apoB. These interactions impact the levels of atherogenic apoB-lipoproteins in the bloodstream.
Understanding this mechanism of hepatic lipoprotein regulation has the potential to inspire novel therapeutic strategies for lowering atherogenic apoB-lipoproteins, ultimately reducing cardiovascular risk in patients. Further research in this area may hold the key to developing innovative treatments for cardiovascular diseases associated with dysregulated lipoprotein metabolism.
reference link: https://www.science.org/doi/10.1126/science.adh5207
