(Page 2 of 2)

Activation of the coagulation cascade generates thrombin. Thrombin is a very efficient proteolytic enzyme—it cleaves other proteins at specific points in their amino acid sequences. One of the proteins it cleaves is fibrinogen, which makes fibrin, the sticky, clot-forming protein. Thrombin also cleaves receptor proteins displayed on cell surfaces, which then "transduce" a signal inside the cell. The first of such a receptor identified was the thrombin receptor or protease activated receptor 1 (PAR1), a receptor of the broad class of G protein-coupled receptors. The activation of this receptor on platelets is critical for platelet activation and hemostasis.

Like all coagulation factors, thrombin circulates as a zymogen—an inactive precursor. If the body needs to make use of thrombin for the clotting cascade, for instance, it must first activate the zymogen "prothrombin" by clipping off the "pro-" part to get thrombin. Obviously, this action must be tightly controlled by the body to avoid causing blood clots willy-nilly. One of the ways the body accomplishes this is by requiring prothrombin to associate with other molecules—called cofactors—before it can be processed into its mature form. The body uses other enzymes to control the cofactors and activated protein C is the enzyme that inactivates the prothrombin cofactor. Activated protein C, thus, in an indirect way controls blood clotting.

Activated protein C is itself normally present as a zymogen, called protein C, and it only becomes active when it is cleaved into its active form. Interestingly, thrombin is the molecule responsible for activating protein C. Thus the two work together in a feedback loop to balance each other, thrombin activating the protein C, which deactivates the cofactors that make thrombin, which reduces the amount of activated protein C, and so on.

This balance is important for maintaining good health and the feedback loop that generates activated protein C occurs on the surface of endothelial cells. Endothelial cells are one of the major cell types of the body, accounting for about one percent of the total cells in the body. They line all blood vessels and capillaries and contribute to the structural integrity of the circulatory system.

Once endothelial cells go, big problems arise—when endothelial cells suffer widespread damage, organs can fail.

The Mystery Solved

Inactive protein C binds to a specific receptor on the surfaces of these endothelial cells—called endothelial cell protein C receptor (EPCR). There, the inactive protein C can be activated by thrombin. To do so, thrombin needs to bind to another endothelial cell receptor, termed thrombomodulin. When thrombin is bound to thrombomodulin, thrombin looses all its clotting function and solely serves to activate the protein C pathway. In sepsis, the physiological balance between thrombin and activated protein C is lost, because inflammatory cytokines cause a loss of thrombomodulin from endothelial cells. Thrombin can no longer activate protein C, and without activated protein C, the endothelial cells cannot be protected.

Ruf and his colleagues have now shown that ECPR is also required for activated protein C to trigger a cascade that upregulate genes that prevent apoptosis, or programmed cell death, and genes that downregulate inflammation and protect these endothelial cells from damage.

Surprisingly, this cascade involves the thrombin receptor PAR1. PAR1 was the missing link between the activated protein C and its protective effect on endothelial cells. Clinical trials had established that endothelial cells can be protected by activated protein C during sepsis, but nobody knew how.

Ruf and his colleagues demonstrated that activated protein C protected these endothelial cells through PAR1 signaling by asking whether the genes that the activated protein C induced could be accounted for by the activation of the PAR1 receptor.

They ran gene profiling using gene chip microarrays, which allowed them to look on a genome-wide scale, and they found about 100 genes that are reproducibly turned on by PAR1. Then they compared those to all the protective genes that are upregulated by activated protein C, and they found that they could all be accounted for by the activation of the PAR1 receptor.

Thus, the mystery was solved. Thrombin binds to thrombomodulin on the surface of endothelial cells and activates the nearby protein C bound to the EPCR. And activated protein C will then activate the PAR1 receptor.

However, Ruf and his colleagues also found a few genes that were upregulated by PAR1 signaling, but not by the signaling of activated protein C. Some of these genes may explain why thrombin has inflammatory effects, while activated protein C is protective and beneficial. One current direction of the laboratory is to determine what combinations of gene upregulations confer protection or cause damage of endothelial cells

The article, "Activation of Endothelial Cell Protease Activated Receptor 1 by the Protein C Pathway" was authored by Matthias Riewald, Ramona J.Petrovan, Aaron Donner, Barbara M. Mueller, and Wolfram Ruf and appeared in the June 7, 2002 issue of the journal Science.


1 | 2 |


When Endothelial Cells See Thrombin

In the top panel, inactive protein C (pink) associates with the EPCR receptor (blue) on the surface of an endothelial cell. Thrombin (purple), which is drawn to thrombomodulin (green), activates protein C. The consequence of this, shown in the bottom panel, is that activated protein C will then activate the PAR1 receptor.