News and Publications

Division of Experimental Hemostasis and Thrombosis

Zaverio M. Ruggeri, M.D., Division Head

Hemostasis is the process that stops bleeding from wounds; it prevents blood from flowing outside the vascular bed. Thrombosis is an abnormal condition in which the vascular lumen becomes occluded by a mass that consists primarily of aggregated platelets and fibrin. These thrombi impede the normal flow of circulating blood, causing diseases that are some of the greatest health concerns in the developed world.

Hemostasis and thrombosis are two aspects of the same function, namely the biological response to vascular injury leading to formation of a thrombus. These two processes, however, lead to ultimate results of opposite value for living organisms. In one, the outcome is the beneficial sealing of a traumatic rupture in a vessel, thus preventing blood loss and eventually death by exsanguination. In the other, the outcome is the damaging sudden occlusion of an artery supplying blood to tissues, causing irreversible cellular alterations and potentially deadly organ failure.

In mechanistic terms, there appears to be little, if any, difference in the initiation and progression of thrombus formation induced by (1) a traumatic vascular lesion, as in a wound, or (2) the sudden instability of a chronic pathologic lesion in the arterial wall, as in plaque rupture in the course of atherosclerosis. Nevertheless, genetic and biochemical markers exist that can be used to determine persons in whom the tendency of blood to form clots in response to either physiologic or pathologic stimuli most likely is enhanced. One aspect of the work performed by investigators in the Division of Experimental Hemostasis and Thrombosis is to characterize and define the predictive value of such markers, ultimately to allow better determination of patients at risk for cardiovascular diseases. Moreover, major efforts are devoted to understanding the intimate mechanistic details of how blood clots are formed and how the development of clots can be controlled. The goal is to advance our understanding of fundamental biological processes and find better therapeutic approaches to the prevention and treatment of myocardial infarction, stroke, and other thromboembolic diseases.

Specific Synergy of Multiple Substrate-Receptor Interactions in Platelet Thrombus Formation Under Flow

B. Savage, F. Almus-Jacobs, Z.M. Ruggeri

We used real-time confocal videomicroscopy to delineate the adhesive interactions that support formation of platelet thrombus on biologically relevant surfaces. Type I collagen fibrils exposed to flowing blood adsorb von Willebrand factor, to which platelets become initially tethered with continuous surface translocation mediated by the membrane glycoprotein Ib. This step is essential at high wall shear rates to allow subsequent irreversible adhesion and thrombus growth mediated by the integrins ₂ß₁ and _IIbß₃ (the latter is also known as platelet glycoprotein IIb-IIIa complex). On subendothelial matrix, endogenous von Willebrand factor and adsorbed plasma von Willebrand factor synergistically initiate platelet recruitment, and ₂ß₁ remains key, along with _IIbß₃, for normal thrombus development at all but low shear rates. Thus, hemodynamic forces and substrate characteristics define the platelet adhesion pathways leading to thrombogenesis.

Contribution of Distinct Adhesive Interactions to Platelet Aggregation in Flowing Blood

Z.M. Ruggeri, J.A. Dent, E. Saldívar

Hemostasis, the vital process that controls hemorrhage, depends on the aggregation of circulating blood platelets onto subendothelial and extravascular substrates exposed at wound sites, particularly when bleeding involves arterioles where rapid flow creates high wall shear rates. Atherosclerotic lesions that restrict the vascular lumen, for example, in coronary arteries, also generate elevated shear rates and may promote the formation of platelet-rich thrombi that block blood flow, causing life-threatening organ damage. The bonds that link platelets to one another to oppose hemodynamic forces during these physiopathologic events are still undefined.

To address this problem, we implemented an original method for the 3-dimensional quantitative evaluation in real time of platelet aggregation in flowing blood. We used surface-immobilized collagen type I fibrils as a model thrombogenic substrate. We found that glycoprotein Ib and von Willebrand factor act in synergy with integrin _IIbß₃ and fibrinogen to sustain platelet accrual at the apex of thrombi, where 3-dimensional growth results in increasing shear rates. This novel model of platelet aggregation highlights the distinct roles of specific adhesion pathways in response to changing hemodynamic forces.

Enhanced Shear-Induced Platelet Aggregation in Acute Myocardial Infarction

S. Goto, H. Sakai, M. Goto, M. Ono, Y. Ikeda, S. Handa, Z.M. Ruggeri

Experimental evidence obtained with models of platelet function under controlled flow conditions indicates that the synergistic interaction of plasma von Willebrand factor (vWF) with glycoproteins Ib and _IIbß₃ is crucial for platelet aggregation and thrombus formation at elevated shear rates. We have now tested how the plasma of patients with acute myocardial infarction affects vWF-dependent shear-induced platelet aggregation. Citrated plasma was obtained from 18 patients with acute myocardial infarction within 6 hours from the onset of signs and symptoms and from 26 control subjects with chest pain syndrome without evidence of ischemia. Normal platelets obtained from healthy donors were mixed with samples of plasma from either control subjects or patients, and aggregation of the platelets was measured with a modified cone-and-plate viscometer and correlated with the plasma levels of vWF antigen and ristocetin cofactor activity.

The extent of platelet aggregation was greater with plasma from patients with myocardial infarction than with plasma from control subjects at the higher shear rates tested. The observed values were positively correlated with plasma vWF antigen levels and ristocetin cofactor activities. Aggregation was markedly inhibited by monoclonal antibodies that blocked the function of the glycoproteins Ib and _IIbß₃. In contrast, aggregation occurring at the lower shear rate of 1200 sec^­1 was essentially the same in the presence of either plasma from control subjects or plasma from patients with myocardial infarction and was not inhibited by the antibodies to glycoprotein Ib. Both vWF antigen and platelet aggregation decreased 2 weeks after the onset of myocardial infarction.

Shear-induced platelet aggregation is enhanced in the presence of plasma from patients with acute myocardial infarction, apparently as a result of increased plasma concentrations of vWF. This mechanism may contribute to the onset of acute coronary artery thrombosis and early reocclusion after reperfusion treatment of such patients.

Structure and Function of Adhesion Molecules

R. Celikel, R. McClintock, B. Gutierrez, P. Marchese, J.R. Roberts, M.L. Thorn, S. Russel, K.I. Varughese, S. Vasudevan, Z.M. Ruggeri

A considerable effort is under way to define at the atomic level the structure of the adhesive receptors and ligands responsible for initiating and propagating the formation of platelet thrombus. After solving the A1 domain of von Willebrand factor (vWF), we are focusing on expressing in mammalian cells a dimeric fragment that contains all 3 contiguous type A domains of the factor. Our intent is to elucidate the structure of a mutifunctional region of vWF that is key for interactions of vWF with the subendothelium and platelets.

Moreover, to understand how conformational changes regulate the affinity of vWF for the glycoprotein Ib, we have started crystallization trials of A1 domain molecules with single-point mutations. We are also attempting to crystallize the amino-terminal domain of glycoprotein Ib itself, which contains binding sites for the vWF A1 domain and the agonist -thrombin. Our aim is to obtain the atomic structures of vWF domains in complex with glycoprotein Ib and with collagen fragments. These structures should provide the definitive information required to understand key interactions leading to thrombus formation and help in the design of specific inhibitors as new therapeutic antithrombotic agents.

PUBLICATIONS

Celikel, R., Varughese, K.I., Madhusudan, Yoshioka, A., Ware, J., Ruggeri, Z.M. Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab. Nature Struct. Biol. 5:189, 1998.

Goto, S., Ikeda, Y., Saldívar, E., Ruggeri, Z.M. Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J. Clin. Invest. 101:479, 1998.

Kritzik, M., Savage, B., Nugent, D.J., Santoso, S., Ruggeri, Z.M., Kunicki, T.J. Nucleotide polymorphisms in the ₂ gene define multiple alleles which are associated with differences in platelet ₂ß₁ density. Blood 92:2382, 1998.

Mazzucato, M., De Marco, L., Masotti, A., Pradella, P., Bahou, W.F., Ruggeri, Z.M. Characterization of the initial -thrombin interaction with glycoprotein Ib in relation to platelet activation. J. Biol. Chem. 273:1880, 1998.

Savage, B., Almus-Jacobs, F., Ruggeri, Z.M. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 94:657, 1998.

Ware, J., Russell, S., Ruggeri, Z.M. Cloning of the murine glycoprotein Ib gene highlighting species-specific platelet adhesion. Blood Cells Mol. Dis. 23:292, 1997.