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Mechanisms of Hemostasis
Summary: Evan Sadler studies the structure, function, and regulation of hemostatic adhesive proteins and proteases.
Blood clots occur normally only at sites of vascular injury, and unnecessary clots are dissolved promptly. Inappropriate blood clots cause devastating illness, such as stroke and heart attack. Thrombosis also complicates many other common diseases, including cancers and infections, and the risk of thrombosis increases as we age.
In the blood, proteins and small cells called platelets are required for clot formation. The endothelial cells that line all blood vessels and circulating white blood cells, however, are not passive bystanders in these reactions but actively promote or inhibit clotting. Compounds that are produced during inflammation also modulate these cellular activities.
We investigate the regulation, structure, and function of proteins that control blood coagulation. My goal is to understand how these opposing tendencies—to stimulate or to inhibit clotting—are balanced to achieve normal hemostasis. These studies may indicate new approaches to treating bleeding and thrombosis.
von Willebrand Factor and von Willebrand Disease
The von Willebrand factor (VWF) is a blood protein that is made by endothelial cells and is required for normal platelet function. VWF binds to and stabilizes blood coagulation factor VIII, the protein that is deficient in classical hemophilia. Hereditary deficiency of VWF, or von Willebrand disease (VWD), is a common genetic bleeding disorder of humans. Symptomatic VWD affects approximately 100 per million persons, and at least 0.2 percent of the population are asymptomatic heterozygous carriers of VWD mutations.
The assembly of VWF is a complex process. The pro-VWF subunit contains five kinds of structural domains in the order D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK. After translocation into the endoplasmic reticulum, pro-VWF dimerizes through disulfide bonds between carboxyl-terminal "cystine knot" (CK) domains. The pro-VWF dimers are transported to the Golgi complex where the propeptide (consisting of domains D1-D2) is removed and additional disulfide bonds are formed between D3 domains near the amino termini of the mature subunits. The product is an enormous multimeric protein that typically contains more than 40 subunits and can be longer than 4 micrometers, which is half the diameter of a red blood cell. A variety of mutations can disrupt this assembly process and cause VWD.
The Structure of VWF Multimers
We have characterized several critical disulfide bonds that link VWF subunits into multimers, and their locations indicate similarities between VWF and other proteins that are assembled intracellularly. VWF subunits associate "tail-to-tail" through their CK domains, which are homologous to members of the transforming growth factor β (TGFβ) family. These growth factors usually are dimeric, and many proteins use related CK motifs to mediate dimerization. Analysis of recombinant VWF CK domains shows they share three intrachain disulfide bonds with other members of the TGFβ family: bonds between cysteines 2–5 and cysteines 3–6 define a ring that is penetrated by a disulfide bond between cysteines 1–4. This knot-like arrangement is responsible for the name "cystine knot." Dimerization is mediated by additional cysteines that differ among CK-domain subfamilies. In the case of VWF, either all or only one of the residues Cys2771, Cys2773, and Cys2811 contributes to intersubunit disulfide bonds.
VWF subunits also associate "head-to-head" through disulfide bonds between their D3 domains. These bonds do not form in the endoplasmic reticulum where disulfides usually are made. Instead, the VWF propeptide acts as an endogenous chaperone to facilitate disulfide bond formation in the relatively hostile environment of the Golgi complex. Our recent mass spectrometry studies identify two interchain disulfide bonds between D3 domains. Inspection of gene sequences from organisms as diverse as insects, roundworms, and vertebrates shows the same strategy of dimerization through carboxyl-terminal CK domains, followed by multimerization through amino-terminal D1-D2-D'-D3 domains, appears to be a common solution to the problem of how to assemble truly gigantic proteins inside a cell. (Our studies of VWF multimer assembly and structure are supported in part by a grant from the National Heart, Lung, and Blood Institute.)
VWF Proteolysis and Thrombotic Thrombocytopenic Purpura
Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, often accompanied by neurological dysfunction, renal failure, and fever. If untreated, the mortality exceeds 90 percent, but plasma exchange therapy has reduced the mortality to less than 20 percent. TTP often strikes young women, suggesting an autoimmune etiology. In fact, most adults with TTP have acquired autoantibodies that inhibit a VWF-cleaving protease in normal blood plasma. Cleavage is stimulated by fluid shear stress that occurs at sites of VWF-dependent platelet adhesion, and this cleavage limits the growth of platelet-rich microvascular thrombi. Deficiency of the protease allows thrombosis to proceed unchecked, causing tissue injury and, eventually, death.
The VWF-cleaving protease recently was identified as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. We determined the structure of the ADAMTS13 gene, as did David Ginsburg (HHMI, University of Michigan) by an independent approach that showed mutations in the ADAMTS13 gene cause an inherited form of TTP.
In addition to its metalloprotease domain, ADAMTS13 has several unusual structural motifs, including a disintegrin-like domain, a thrombospondin repeat, a cysteine-rich domain, a spacer domain, seven more thrombospondin repeats, and two CUB domains. We have identified conditions that allow ADAMTS13 to bind VWF with high affinity and cleave it, and these activities depend on the first thrombospondin repeat and the spacer domain of ADAMTS13. Surprisingly, the seven carboxyl-terminal thrombospondin repeats and the CUB domains seem to contribute little to the recognition of VWF. These additional carboxyl-terminal domains are conserved among ADAMTS13 proteases from humans, other mammals, birds, amphibians, and fish. Such structural stability throughout evolution suggests that these carboxyl-terminal domains have important functions that we do not yet understand, although our preliminary data suggest the CUB domains can sometimes regulate binding to VWF.
ADAMTS13 cleaves a specific peptide bond in the VWF A2 domain. The adjacent A1 and A3 domains bind to platelet glycoprotein Ibα (GPIbα) and collagen, respectively, and this arrangement suggests that interactions of VWF with platelets or collagen might also influence its susceptibility to ADAMTS13. We tested this with recombinant substrates derived from VWF domains A1A2A3. Deletion of domain A3 did not affect cleavage by ADAMTS13, but deletion of domain A1 increased the rate of cleavage about 10-fold. Cleavage of A1A2A3 also was enhanced by a recombinant fragment of platelet GPIbα or by heparin, each of which binds to domain A1. The results suggest that VWF domain A1 inhibits the cleavage of domain A2, and that inhibition can be relieved by interaction of domain A1 with platelets or with glycosaminoglycans such as heparin. Thus, binding of VWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion, by stimulating the cleavage of VWF by ADAMTS13.
In recent clinical studies, we found that patients with TTP who have normal ADAMTS13 levels have a very poor prognosis. Conversely, TTP caused by severe ADAMTS13 deficiency tends to respond well to treatment with plasma exchange, although the presence of autoantibody inhibitors of ADAMTS13 correlates with a high likelihood of relapsing disease. In some patients, intensive immunosuppressive therapy has been able to stop the production of ADAMTS13 inhibitors and produce a lasting remission from TTP. We are continuing to study the value of ADAMTS13 data for the diagnosis and treatment of TTP in further clinical trials.
Last updated January 24, 2007
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