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Rap1b: Wrapping up Blood Clotting Abstract Blood clotting is crucial for maintaining homeostasis, or equilibrium of internal conditions. However, unintended consequences may result if blood is unable to clot or clots excessively. The protein Rap1b plays a key role in the process of regulating blood clotting, which is facilitated by platelets sending activation signals. When endothelial cells are damaged, matrix proteins are exposed; hence, activation signals are sent to Rap1b. In its inactive state, Rap1b is bound to GDP. Through the replacement of GDP with GTP, two switch regions on the now-activated Rap1b change shape. With the help of guanine nucleotide exchange factors (GEFs) and GAP proteins, Rap1b then binds to an effector protein, activating integrins, which control the attachment of cells to matrix proteins. In turn, the activated integrins located on the cell membrane of a platelet allow platelets to stick together, forming a blood clot. Research has shown that if a mutation occurs at amino acid N17, the protein is permanently set to an inactive state. However, when amino acid G12 mutates into valine, Rap1b is constitutively active. An imbalance in the signaling cascade of blood clotting leads to health risks, such as strokes or bleeding disorders. Studying Rap1b brings us one step closer to understanding how the body regulates platelet activation and clotting. Whitefish Bay SMART Team Students: Colleen Ackermann, Isaiah Kaplan, Lazura Krasteva, Shannon Mahony, Andrew Phillips, Zack Serebin and Alice Xia Teachers: Paula Krukar and Michael Krack Mentors: Jackie Porath and Gil White, MD, Blood Research Institute, Blood Center of Wisconsin Cell Signaling Chain Leading to Platelet Aggregation Step 1: Damage to blood vessel exposes matrix proteins Step 2: Receptor on platelet detects matrix proteins and binds Step 3: PLC generates messenger molecules, Ca+2 and DAG Ca+2 Introduction In this graph, the wild type (WT) mice, with Rap1b, had a significantly lower bleeding time, while the knock-out (KO) mice had an extended bleeding time. This can be caused by either leaky blood vessels or dysfunctional platelets, specifically due to a lack of Rap1b. A bone marrow transplant experiment was then conducted. The WT mice with WT bone marrow experienced a shorter bleeding time, whereas the WT mice with KO bone marrow experienced a longer bleeding time. Thus, the increased bleeding time suggests that the problem was in fact the dysfunctional platelets. Since the platelets were Rap1b deficient, Rap1b is needed for proper platelet function. Step 4: Activation of Rap activator, Cal-DAG GEF DAG When damage occurs to capillaries and blood vessels, platelets within the blood clot to stop the bleeding. In this case, the coagulation of blood around the site of injury is beneficial to the body. However, blood clotting can occur unnecessarily, forming dangerous clots that can result in heart attack or stroke. Rap1b, a protein found inside the membrane of platelets, plays a crucial role in facilitating this blood coagulation. When activated, Rap1b signals platelets to stick together, which results in a blood clot. Rap1b: Necessary for Clotting Step 5: GDP is removed by Cal-DAG GEF and GTP is added, changing Rap1b from inactive to active. Biological Significance GTP GDP GTP GDP Inactive Inactive Active Rap1b Rap1b Rap1b Step 6: Integrin activation Mutations Inactive Integrin Rap1b has two significant mutations. When the Glycine 12 amino acid is exchanged for valine, Rap1b permanently switches on. Additionally, when the Serine 17 amino acid is changed to asparagine, Rap1b becomes permanently inactive. These mutations can cause either excess clotting or lack of clotting, respectively. Active Integrin Active Integrin Fibrinogen Step 7: Integrins from two different platelets bind to fibrinogen PDB File: 3BRW Blood clotting caused by platelets can have both life-saving and lifethreatening effects on the human body. In platelets, Rap1b, a membrane protein, is essential to the clotting of blood. Rap1b switches from an inactive to an active state when matrix proteins are detected. Without Rap1b, platelets do not clot. Currently, scientists are trying to connect the discoveries of Rap1b to the Ras proteins, which, when mutated to a permanently active state, cause over-proliferation of cells, resulting in cancer. With the knowledge gained from Rap1b, scientists can hopefully prevent this mutation and thus prevent some types of cancer. Step 8: Platelet aggregation causes a blood clot to form A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA) and an NIH CTSA Award to the Medical College of Wisconsin. . Platelet Platelet © Health and Life References: The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues. Scrima, A., Thomas, C., Deaconescu, D., Wittinghofer, A. Journal: (2008) Embo J. 27: 1145-1153 Rap1b is required for normal platelet function and hemostasis in mice. Magdalena ChrzanowskaWodnicka, Susan S. Smyth, Simone M. Schoenwaelder, Thomas H. Fischer, and Gilbert C. White II.