Hemostasis and thrombosis, sickle cell anemia, TTP, ITP
Platelet adhesion and von Willebrand factor, platelets in inflammation, sickle cell vasooclusion
Platelet adhesion and von Willebrand factor functions: The laboratory has been involved for many years in studies aimed at understanding the basic mechanisms of platelet adhesion. Initially, these studies were aimed at the synthesis, assembly, posttranslational modifications and defining the ligand-binding sites of the receptor that mediates the first step of platelet adhesion, the glycoprotein (GP) Ib-IX-V complex. These studies are continuing, now focused on the association of the GP Ib-IX-V complex with membrane microdomains known as lipid rafts and downstream signaling after the complex has bound its primary adhesive ligand, von Willebrand factor (VWF), a multimeric polypeptide synthesized in endothelial cells and megakaryocytes.
The work also extends into the functions of VWF. Work from the laboratory over the past several years has demonstrated that when endothelial cells are stimulated to release VWF-containing granules, the Weibel-Palade bodies, VWF forms extremely long strings on the endothelial surface, to which platelets rapidly attach. In the presence of plasma, the strings are rapidly cleaved by the action of the metalloprotease ADAMTS-13. In contrast, plasma from patients afflicted with the systemic microthrombotic disorder, thrombotic thrombocytopenic purpura (TTP), fails to cleave the strings, providing a mechanism for microvascular occlusions seen in the disorder. Because of their extraordinarily large size, newly released VWF is called ulta-large VWF, or ULVWF. We have also shown that ULVWF is not only enormous; it is also more adhesive at the single bond level. The molecular basis of this increased reactivity is a major topic of research in the laboratory.
The interactions of platelets with endothelium and leukocytes: A considerable amount of recent research indicates that platelets play roles in inflammation that goes beyond their hemostatic role. We have contributed to this work. We have shown, for example, that lowering the platelet count with anti-platelet antibodies markedly protects mice from the systemic inflammatory response that accompanies resuscitation from hemorrhagic shock by effects that include the diminution of neutrophil infiltration. We have begun to dissect the molecular basis of the effect by defining counter-receptors on platelets for neutrophil integrins. We found that leukocyte Mac-1 (aMb2) binds platelet GP Iba, the largest component of the GP Ib-IX-V complex and the polypeptide responsible for binding VWF. The interaction of GP Iba with Mac-1 is of particular interest, as we have recently shown that blocking it prevents restenosis after vessel injury and profoundly reduces inflammation after corneal injury. We are exploring the possibility of developing blocking agents to treat inflammatory disorders.
Adhesive interactions in sickle cell vaso-occlusion: Work in our laboratory has focused on the role of the sulfated glycolipids sulfatide as playing an important role in sickle cell vaso-occlusion. We have produced a single-chain variable fragment antibody that recognizes sulfatide and shown that erythrocytes from sickle cell patients express more of the lipid on their surfaces. Sickle and normal erythrocytes adhere to surfaces of VWF, thrombospondin-1, and laminin, with adhesion of sickle cells being more resistant to the effects of shear stress. This adhesion is blocked by the anti-sulfatide antibody and is reduced considerably in mice incapable of synthesizing sulfatide. As VWF is appears to be an important adhesive receptor for sickle cells, we examined whether the decreased activity of ADAMTS-13, which not only cleaves ULVWF but also process it to less adhesive forms, might predispose a patient to developing vaso-occlusion. Our preliminary data indicates that this is the case. We are continuing this work.
Platelet, leukocyte and endothelial microparticles in health and disease: One of the projects in the laboratory concerns membrane blebs called microvesicles or microparticles and their roles in normal hemostasis, thrombosis, and acute inflammatory responses. Our data indicate that microparticles play important roles in endotoxin-induced disseminated intravascular coagulation. We are now examining their roles in venous thrombosis.
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Microparticles and thrombotic disease.
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