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4.W25.2 Organization of apolipoprotein A-I domains in solution and in reconstituted high density lipoproteins
Thursday 9 October 1997: Workshops W25 Molecular insights into HDL and its properties by both protein tyrosine kinases (PTK) and phosphatases (PTP) . In platelets P plays a key role in signal transduction. These events seem to be programmed to occur in a temporal order throughout the whole process of platelet activation : shape change, release, aggregation, clot retractation . Thrombin treatment causes a dramatic increase in the level of phosphotyrosines that have been shown to be either alIb/143 and aggregation "independent" or "dependent" . In Glanzmann thrombasthenia, in which platelets do not aggregate, tyrosine P of the 100-105 kDa doublet as well as the 125 kDa (FAK) is defective. However the 77/80 kDa doublet identified as the cytoskeletal protein, cortactin undergoes a normal early P Cortactin is then associated to early platelet activation in parallel with shape change and the formation of filopods dependent upon cytoskeletal arrangement. Thrombin stimulation leads to the activation of Pi3 kinase (Pi3K) which depends on aIIb1143 engagement and requires tyrosine P for its translocation to the actin skeleton and the production of Pi3,4P2 . Pi3K and Pi3,4P2 are then associated to late events of platelet activation dependent on fibrinogen binding to allb/$3 and aggregation . Finally extracellular-signal regulated kinase (ERK) activation seems to be related to post aggregation events since RGDS does not inhibit either ERK2 phosphorylation or activity . Moreover the inhibition or the absence of aIlb/$3 engagement leads to an increase of ERK2 activation suggesting that ERK2 is down regulated by allb/143 . Tyrosine P is necessary for platelet activation but tyrosine dephosphorylation is a prerequisite to avoid platelet activation . Peroxovanadate, a PTP inhibitor, induces platelet aggregation accompanied by a marked increase in tyrosyl P. The regulation and the role of one of the PTP, SH-PTPI will be discussed. The concerted activities of PTK and PTP, tightly dependent from thrombin and integrin receptors, allow to achieve the orchestrated signaling leading to platelet aggregation .
4 .W24 .4
Respective role of GPIb and GPIIb-IIIa in flow
Jan J . Sixma, Ph. G. de Groot, T. Vink, H . Lankhof, Yaping Wu . Dept of Haematology, University Hospital Utrecht, The Netherlands Von Willebrand Factor is an essential adhesive protein present in platelets, subendothelium and in plasma . Studies by others with washed platelets have indicated that GPIb serves as a rolling receptor whereas GPIIb-IIIa is responsible for firm attachment. We studied the relative contribution of these 2 types of platelet receptor using mutant recombinant vWF which was either defective in the interaction with GPIb because the Al-domain had been deleted or with GPIIb-IIIa because the RGDS -sequence had been changed to RGGS . We found that even under static conditions GPIb is responsible for adhesion whereas GPIIb-IIIA is required for spreading . Under flow both vWF receptor interactions where required when vWF was the only adhesive protein . When vWF was adsorbed to collagen type I or III, GPIb was the essential receptor, but some decrease in adhesion was observed with RGGS-vWF and platelets did not spread well on collagen . At high shear rates, vWF is an essential cofactor for adhesion of platelets to either fibrinogen or fibronectin . In both instances adhesion was mediated by the GPIb/vWF interaction and not by the GPIIb/IIIa interaction . VWF has been shown to cause platelet aggregate formation at high shear rates . In PRP using very high shear rates, platelet GPIb is responsible for aggregation mediated by vWF and activation of platelets occurs. In the aggregate formation of platelets on top of adhered platelets in perfusion with whole blood over collagen at shear rates of 2600/sec, GPIIb-IIIa was held responsible . We investigated this in detail using blood of patients with severe von Willebrands disease to which the above mentioned mutants had been added . Normal adhesion had been ensured by preincubation of surface coated collagen type III with plasma vWF . We found that the GPIb/vWF interaction was responsible for thrombus formation whereas the GPIIb-IIIa interaction was inhibitory probably because of competition with other plasma ligands such as fibrinogen and/or fibronectin . This became clear when the RGGS mutant gave larger thrombi than wild type vWF in vWD-blood but not in reconstituted blood with only human albumin as other plasma protein apart from vWF . We conclude that the RGD sequence in vWF has a minor role in platelet adhesion and none or an inhibitory one in thrombus formation . The RGD-sequence is probably more important in the adhesion of endothelial cells .
4 .W24.5
Molecular requirements for ligand recognition by 03 integrins
T.J . Kunicki. The Scripps Research Institute, La Jolla CA 92037, USA With proteins that bind to integrins via the tripeptide motif Arginine-GlycineAspartate (RGD), integrin specificity is modulated by subtle changes in the
293
juxtaposition of the Arginine (Arg) and Aspartate (Asp) side chains . Insight has been obtained from a comparison of the binding properties of the recombinant monoclonal Fab molecules of the AP7 series . These Fab molecules, which contain the RGD sequence in CDR3 of their respective H chains (H3), represent a valuable paradigm of natural RGD ligands . To wit, the amino acid composition immediately adjacent to the RGD tripeptide can change the specificity of the ligand for 03 integrins . Thus, AP7 (HPFYRGDGGN) binds exclusively to aHb133, while AP7 .4 (HPFYRGDGGA) binds solely to av$3 . Our results would argue that, in comparison to avs3, a ll 3 is more selective with respect to the Arg and Asp side chain orientations that it will recognize . Affinity modulation, too, is governed by subtle differences in structure within the immediate environment of the RGD sequence . We compared the binding of AP7 to that of the engineered variant, AP7.3, in which the H3 loop sequence of AP7 was replaced by that of the activation-dependent Fab molecule (RSPSYYRGDGAGP) . AP7 does not bind preferentially to activated albf3, but AP7 .3 does . The fact that AP7 .3 behaves identically, in parallel binding experiments, to the prototype activation-dependent ligand PAC 1 .1 argues that a very subtle change in the position and/or orientation of the RGD sequence in this CDR loop is sufficient to modify the dependence of ligand binding on the conformational state of the receptor.
W25
4.W25 .1
MOLECULAR INSIGHTS INTO HDL AND ITS PROPERTIES Anti-atherogenic properties of HDL : Insights from the A-IMiiano mutation
G . Franceschini . Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milano, Italy The apolipoprotein A-IMIh„o (A-IM) mutant is characterized by the Arg 173 - . Cys substitution, leading to the formation of disulfide-linked homodimers (A-IM/A-IM) and heterodimers with apoA-ll . Carriers have low plasma HDIL levels but no increased risk of premature vascular disease . The A-IM/A-IM has been expressed in E . coli and used as a tool to investigate the anti-atherogenic properties of apoA-I . The introduction of the disulfide bridge remarkably limits the conformational flexibility of the protein when incorporated into reconstituted HDL (rHDL) . In vitro studies with various cell types indicate thatt apoA-I functions are drastically and differently affected by this structural change . The ability to promote cell cholesterol efflux and stimulate fibrinolysis are increased, while LCAT activation is impaired . The HDL-mediated inhibition of adhesion molecules expression on endothelial cells is not affected. When infused into cholesterol-fed rabbits, A-IM/A-IM-containing rHDL are more effective than those with apoA-I in preventing the formation of a vascular lesion following periarterial manipulation . The A-IM variant proved to be a useful tool to understand structure-function relationships in apoA-I and to design novel apoA-I mutants with improved potential in the prevention and treatment o1' arterial disease.
4 .W25 .2
Organization of apolipoprotein A-I domains in solution and in reconstituted high density lipoproteins
A . Jonas, W.S . Davidson . University of Illinois at Urbana-Champaign, Urbana, IL, USA The key function of apolipoprotein A-I (apoA-I) is lipid binding and high density lipcprotein (HDL) stabilization . Other functions of apoA-I depend on its primary interaction with phospholipids and include cholesterol transport, lecithin-cholesterol acyltransferase (LCAT) activation, interaction with lipid transfer proteins, and recognition of cell receptors . Our work has focused on the interaction of apoA-I with phospholipids, on the conformations that apoA-I adopts in this process, and on the domains of apoA-I involved in its functions . Using site directed (Trp -a Phe) and deletion mutants of apoA-I, as well as a variety of biophysical and biochemical approaches, we have shown that in aqueous solution apoA-I has substantial 2-dimensional structure in the N-terminal domain while the C-terminal region is disorganized . The initial interaction with phospholipid bilayers involves the extreme C-terminus of the molecule and specific N-terminal Trp residues . Subsequent stabilization of HDL structure requires the development of C-terminal a-helices and the reorganization of the 2-dimensional structure at the N-terminus . In addition, we have used computer modeling of HDL and molecular dynamics to gain further insights into the end-to-end contacts of apoA-I molecules and their overall organization. (Supported by NIH grant HL-16059 .)
11th International Symposium on Atherosclerosis, Paris, October 1997
4 .W24 .4
Respective role of GPIb and GPIIb-IIIa in flow
Jan J . Sixma, Ph. G. de Groot, T. Vink, H . Lankhof, Yaping Wu . Dept of Haematology, University Hospital Utrecht, The Netherlands Von Willebrand Factor is an essential adhesive protein present in platelets, subendothelium and in plasma . Studies by others with washed platelets have indicated that GPIb serves as a rolling receptor whereas GPIIb-IIIa is responsible for firm attachment. We studied the relative contribution of these 2 types of platelet receptor using mutant recombinant vWF which was either defective in the interaction with GPIb because the Al-domain had been deleted or with GPIIb-IIIa because the RGDS -sequence had been changed to RGGS . We found that even under static conditions GPIb is responsible for adhesion whereas GPIIb-IIIA is required for spreading . Under flow both vWF receptor interactions where required when vWF was the only adhesive protein . When vWF was adsorbed to collagen type I or III, GPIb was the essential receptor, but some decrease in adhesion was observed with RGGS-vWF and platelets did not spread well on collagen . At high shear rates, vWF is an essential cofactor for adhesion of platelets to either fibrinogen or fibronectin . In both instances adhesion was mediated by the GPIb/vWF interaction and not by the GPIIb/IIIa interaction . VWF has been shown to cause platelet aggregate formation at high shear rates . In PRP using very high shear rates, platelet GPIb is responsible for aggregation mediated by vWF and activation of platelets occurs. In the aggregate formation of platelets on top of adhered platelets in perfusion with whole blood over collagen at shear rates of 2600/sec, GPIIb-IIIa was held responsible . We investigated this in detail using blood of patients with severe von Willebrands disease to which the above mentioned mutants had been added . Normal adhesion had been ensured by preincubation of surface coated collagen type III with plasma vWF . We found that the GPIb/vWF interaction was responsible for thrombus formation whereas the GPIIb-IIIa interaction was inhibitory probably because of competition with other plasma ligands such as fibrinogen and/or fibronectin . This became clear when the RGGS mutant gave larger thrombi than wild type vWF in vWD-blood but not in reconstituted blood with only human albumin as other plasma protein apart from vWF . We conclude that the RGD sequence in vWF has a minor role in platelet adhesion and none or an inhibitory one in thrombus formation . The RGD-sequence is probably more important in the adhesion of endothelial cells .
4 .W24.5
Molecular requirements for ligand recognition by 03 integrins
T.J . Kunicki. The Scripps Research Institute, La Jolla CA 92037, USA With proteins that bind to integrins via the tripeptide motif Arginine-GlycineAspartate (RGD), integrin specificity is modulated by subtle changes in the
293
juxtaposition of the Arginine (Arg) and Aspartate (Asp) side chains . Insight has been obtained from a comparison of the binding properties of the recombinant monoclonal Fab molecules of the AP7 series . These Fab molecules, which contain the RGD sequence in CDR3 of their respective H chains (H3), represent a valuable paradigm of natural RGD ligands . To wit, the amino acid composition immediately adjacent to the RGD tripeptide can change the specificity of the ligand for 03 integrins . Thus, AP7 (HPFYRGDGGN) binds exclusively to aHb133, while AP7 .4 (HPFYRGDGGA) binds solely to av$3 . Our results would argue that, in comparison to avs3, a ll 3 is more selective with respect to the Arg and Asp side chain orientations that it will recognize . Affinity modulation, too, is governed by subtle differences in structure within the immediate environment of the RGD sequence . We compared the binding of AP7 to that of the engineered variant, AP7.3, in which the H3 loop sequence of AP7 was replaced by that of the activation-dependent Fab molecule (RSPSYYRGDGAGP) . AP7 does not bind preferentially to activated albf3, but AP7 .3 does . The fact that AP7 .3 behaves identically, in parallel binding experiments, to the prototype activation-dependent ligand PAC 1 .1 argues that a very subtle change in the position and/or orientation of the RGD sequence in this CDR loop is sufficient to modify the dependence of ligand binding on the conformational state of the receptor.
W25
4.W25 .1
MOLECULAR INSIGHTS INTO HDL AND ITS PROPERTIES Anti-atherogenic properties of HDL : Insights from the A-IMiiano mutation
G . Franceschini . Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milano, Italy The apolipoprotein A-IMIh„o (A-IM) mutant is characterized by the Arg 173 - . Cys substitution, leading to the formation of disulfide-linked homodimers (A-IM/A-IM) and heterodimers with apoA-ll . Carriers have low plasma HDIL levels but no increased risk of premature vascular disease . The A-IM/A-IM has been expressed in E . coli and used as a tool to investigate the anti-atherogenic properties of apoA-I . The introduction of the disulfide bridge remarkably limits the conformational flexibility of the protein when incorporated into reconstituted HDL (rHDL) . In vitro studies with various cell types indicate thatt apoA-I functions are drastically and differently affected by this structural change . The ability to promote cell cholesterol efflux and stimulate fibrinolysis are increased, while LCAT activation is impaired . The HDL-mediated inhibition of adhesion molecules expression on endothelial cells is not affected. When infused into cholesterol-fed rabbits, A-IM/A-IM-containing rHDL are more effective than those with apoA-I in preventing the formation of a vascular lesion following periarterial manipulation . The A-IM variant proved to be a useful tool to understand structure-function relationships in apoA-I and to design novel apoA-I mutants with improved potential in the prevention and treatment o1' arterial disease.
4 .W25 .2
Organization of apolipoprotein A-I domains in solution and in reconstituted high density lipoproteins
A . Jonas, W.S . Davidson . University of Illinois at Urbana-Champaign, Urbana, IL, USA The key function of apolipoprotein A-I (apoA-I) is lipid binding and high density lipcprotein (HDL) stabilization . Other functions of apoA-I depend on its primary interaction with phospholipids and include cholesterol transport, lecithin-cholesterol acyltransferase (LCAT) activation, interaction with lipid transfer proteins, and recognition of cell receptors . Our work has focused on the interaction of apoA-I with phospholipids, on the conformations that apoA-I adopts in this process, and on the domains of apoA-I involved in its functions . Using site directed (Trp -a Phe) and deletion mutants of apoA-I, as well as a variety of biophysical and biochemical approaches, we have shown that in aqueous solution apoA-I has substantial 2-dimensional structure in the N-terminal domain while the C-terminal region is disorganized . The initial interaction with phospholipid bilayers involves the extreme C-terminus of the molecule and specific N-terminal Trp residues . Subsequent stabilization of HDL structure requires the development of C-terminal a-helices and the reorganization of the 2-dimensional structure at the N-terminus . In addition, we have used computer modeling of HDL and molecular dynamics to gain further insights into the end-to-end contacts of apoA-I molecules and their overall organization. (Supported by NIH grant HL-16059 .)
11th International Symposium on Atherosclerosis, Paris, October 1997