This work is supported by a National Scientist Development grant from the American Heart Association: 10SDG3500034 and UTMB-NCB start up funds to SSM. The authors acknowledge the Cryo-EM and Scientific Computing facilities at the Sealy Center for Structural Biology at UTMB (<a href="http://www.scsb.utmb.edu">www.scsb.utmb.edu</a>), as well as Drs. Steve Ludtke and Ed Egelman for help with the 2D and 3D helical reconstruction algorithms.

1. Sample Preparation
<ol>
	<li>Buffer exchange human FVIII-BDD14 and porcine FVIII-BDD15 against HBS-Ca buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH = 7.4) and concentrate to 1.2 mg/ml. Keep the protein solution at -80 &#176;C.</li>
	<li>Prepare lipid nanotubes (LNT) by mixing GalactosylCeramide (GC) and phosphatidylserine (PS) at 1:4 w/w ratio in chloroform. Evaporate the chloroform under argon and solubilize the lipids in HBS buffer to 1 mg/ml. Keep the LNT solution at 4 &#176;C....

<ol>
	<li>Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Realizing+the+potential+of+electron+cryo-microscopy.">Realizing the potential of electron cryo-microscopy.</a> Quarterly Reviews of Biophysics. 37, 3-13 (2004).</li><li>Fujiyoshi, Y., Unwin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+crystallography+of+proteins+in+membranes.">Electron crystallography of proteins in membranes.</a> Current opinion in structural biology. 18, 587-592 (2008).</li><li>Toole, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+cDNA+encoding+human+antihaemophilic+factor.">Molecular cloning of a cDNA encoding human antihaemophilic factor.</a> Nature. 312, 342-347 (1984).</li><li>Fay, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factor+VIII+structure+and+function.">Factor VIII structure and function.</a> International journal of hematology. 83, 103-108 (2006).</li><li>Stoilova-McPhie, S., Lynch, G. C., Ludtke, S. J., Pettitt, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+membrane-bound+factor+VIII.">Domain organization of membrane-bound factor VIII.</a> Biopolymers. , (2013).</li><li>Pipe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemophilia:+new+protein+therapeutics.">Hemophilia: new protein therapeutics.</a> Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program. 2010, 203-209 (2010).</li><li>Gatti, L., Mannucci, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+porcine+factor+VIII+in+the+management+of+seventeen+patients+with+factor+VIII+antibodies.">Use of porcine factor VIII in the management of seventeen patients with factor VIII antibodies.</a> Thrombosis and haemostasis. 51, 379-384 (1984).</li><li>Parmenter, C. D., Cane, M. C., Zhang, R., Stoilova-McPhie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+coagulation+Factor+VIII+bound+to+lipid+nanotubes.">Cryo-electron microscopy of coagulation Factor VIII bound to lipid nanotubes.</a> Biochemical and biophysical research communications. 366, 288-293 (2008).</li><li>Parmenter, C. D., Stoilova-McPhie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+recombinant+human+coagulation+factor+VIII+to+lipid+nanotubes.">Binding of recombinant human coagulation factor VIII to lipid nanotubes.</a> FEBS letters. 582, 1657-1660 (2008).</li><li>Wassermann, G. E., Olivera-Severo, D., Uberti, A. F., Carlini, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+urease+activates+blood+platelets+through+a+lipoxygenase-mediated+pathway.">Helicobacter pylori urease activates blood platelets through a lipoxygenase-mediated pathway.</a> Journal of cellular and molecular medicine. 14, 2025-2034 (2010).</li><li>Wilson-Kubalek, E. M., Chappie, J. S., Arthur, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helical+crystallization+of+soluble+and+membrane+binding+proteins.">Helical crystallization of soluble and membrane binding proteins.</a> Methods in enzymology. 481, 45-62 (2010).</li><li>Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+helical+filaments+and+tubes.">Reconstruction of helical filaments and tubes.</a> Methods in enzymology. 482, 167-183 (2010).</li><li>Lusher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+introduction+of+recombinant+factor+VIII--a+clinician's+experience.">Development and introduction of recombinant factor VIII--a clinician's experience.</a> Haemophilia : the official journal of the World Federation of Hemophilia. 18, 483-486 (2012).</li><li>Thim, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+a+new+recombinant+factor+VIII+(N8).">Purification and characterization of a new recombinant factor VIII (N8).</a> Haemophilia : the official journal of the World Federation of Hemophilia. 16, 349-359 (2010).</li><li>Doering, C. B., Healey, J. F., Parker, E. T., Barrow, R. T., Lollar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+level+expression+of+recombinant+porcine+coagulation+factor+VIII.">High level expression of recombinant porcine coagulation factor VIII.</a> The Journal of biological chemistry. 277, 38345-38349 (2002).</li><li>Tang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMAN2:+an+extensible+image+processing+suite+for+electron+microscopy.">EMAN2: an extensible image processing suite for electron microscopy.</a> Journal of structural biology. 157, 38-46 (2007).</li><li>Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+algorithm+for+the+reconstruction+of+helical+filaments+using+single-particle+methods.">A robust algorithm for the reconstruction of helical filaments using single-particle methods.</a> Ultramicroscopy. 85, 225-234 (2000).</li><li>Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iterative+helical+real+space+reconstruction+method:+surmounting+the+problems+posed+by+real+polymers.">The iterative helical real space reconstruction method: surmounting the problems posed by real polymers.</a> Journal of structural biology. 157, 83-94 (2007).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> Journal of structural biology. 152, 36-51 (2005).</li><li>Stoilova-McPhie, S., Villoutreix, B. O., Mertens, K., Kemball-Cook, G., Holzenburg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+structure+of+membrane-bound+coagulation+factor+VIII:+modeling+of+the+factor+VIII+heterodimer+within+a+3-dimensional+density+map+derived+by+electron+crystallography.">3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography.</a> Blood. 99, 1215-1223 (2002).</li><li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+density+maps+with+UCSF+Chimera.">Visualizing density maps with UCSF Chimera.</a> Journal of structural biology. 157, 281-287 (2007).</li></ol>

The authors declare that they have no competing financial interest and they can be contacted directly regarding any of the procedures published in this manuscript.

In this work a methodology is presented to differentiate between two membrane-bound organizations of highly homologous proteins: human and porcine FVIII self-assembled on lipid nanotubes in the conditions encountered in the human body.

In the described procedure, human and porcine FVIII are successfully organized helically on lipid nanotubes, which is the most critical step. The next critical step is to preserve the sample in thin amorphous ice by flash freezing&#1...

Blood coagulation Factor VIII (FVIII) is a large glycoprotein of 2,332 amino acids organized in six domains: A1-A2-B-A3-C1-C2&#160;3. Upon Thrombin activation FVIII acts as the cofactor to Factor IXa within the membrane-bound Tenase complex. Binding of activated FVIII (FVIIIa) to FIXa in a membrane-depending manner enhances FIXa proteolytic efficiency more than 105 times, which is critical for efficient blood coagulation4. Despite the important role FVIII plays in coagulation and the Tenase complex formation, the functional membrane-bound FVIII structure is yet to be resolved.
To address this, single lipid bilayer nanotubes (LNT) rich in phosphatidylserine (PS), capable of binding FVIII with high affinity8,9 and resembling the activated platelet surface have been developed10. Consecutive helical organization of FVIII bound to LNT has been proven to be effective for structure determination of FVIII membrane-bound state by Cryo-EM5. Functionalized LNT are an ideal system to study protein-protein and protein-membrane interactions of helically organized membrane-associated proteins by Cryo-EM11,12. Cryo-EM has the advantage over traditional structural methods such&#160;as X-ray crystallography and NMR, as the specimen is preserved at closest to the physiological environment (buffer, membrane, pH), without additives and isotopes. In the case of FVIII, studying the membrane-bound structure with this technique is even more physiologically relevant, as the LNT resemble closely by size, shape and composition the pseudopodia of the activated platelets where the Tenase complexes assemble in vivo.
Defects and deficiency of FVIII cause Hemophilia A, a severe bleeding disorder affecting 1 in 5,000 males of the human population4,6. The most effective therapy for Hemophilia A is life-long administration of recombinant human FVIII (hFVIII). A significant complication of the recombinant FVIII Hemophilia A therapy is the development of inhibitory antibodies to the human form affecting approximately 30% of Hemophilia A patients13. In this case, porcine FVIII (pFVIII) concentrate is used, as porcine FVIII displays low cross-reactivity with inhibitory antibodies against human FVIII and forms functional complexes with human FIXa7. Establishing the membrane-bound organization of both porcine and human FVIII forms is important to understand the structural basis of FVIII cofactor function and implications for blood hemostasis.
In this study, we describe a combination of lipid nanotechnology, Cryo-EM, and structure analysis designed to resolve the membrane-bound organization of two highly homologous FVIII forms. The presented Cryo-EM data and 3D structures for helically organized porcine and human FVIII on negatively charged LNT show the potential of the proposed nanotechnology as basis for structure determination of FVIII and membrane-bound coagulation factors and complexes in a physiological membrane environment.

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">JEM2100 with LaB6&#160;</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;">JEM-2100</td>
			<td style="width:447px;">operated at 200 kV</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">with TEMCON software</td>
			<td style="width:205px;">JEOL Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan626 Cryo-holder</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">626.DH</td>
			<td style="width:447px;">cooled to -175 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">with temperature controler unit</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Gatan 4K x 4K CCD camera</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;">US4000</td>
			<td style="width:447px;">4096 x 4096 pixel at 15 microns/pixel physical resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Solarus Model 950 plasma cleaner</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Vitrobot Mark IV</td>
			<td style="width:205px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Materials</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Carbon coated 300 mesh 3mm copper grid</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">01821</td>
			<td style="width:447px;">plasma cleaned for 10 s on high power</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">Quantifoil R2/2 300 mesh</td>
			<td style="width:205px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Q225-CR2</td>
			<td style="width:447px;">Carbon coated 300 mesh Cu grids with 2 mm in diameters holes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Uranyl acetate dihydrate</td>
			<td style="width:205px;">Ted Pella</td>
			<td style="width:119px;">19481</td>
			<td style="width:447px;">1% solution, filtered</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:239px;">Galactosyl ceramide</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">860546</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dioleoyl-sn-glycero-phospho-L-serine</td>
			<td style="width:205px;">Avanti Polar Lipids Inc.&#160;</td>
			<td style="width:119px;">840035</td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;"></td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Software</td>
			<td style="width:205px;"></td>
			<td style="width:119px;"></td>
			<td style="width:447px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:239px;">EM software Digital Micrograph</td>
			<td style="width:205px;">Gatan, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.gatan.com/DM/">http://www.gatan.com/DM/</a></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">EM software EMAN</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://blake.bcm.edu/emanwiki/EMAN/">http://blake.bcm.edu/emanwiki/EMAN/&#160;</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">EM software Spider</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://spider.wadsworth.org/spider_doc/spider/docs/spider.html">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">EM software IHRSR</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;">Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (IMOD)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">EM software (SerialEM)</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="ftp://bio3d.colorado.edu/pub/SerialEM/">ftp://bio3d.colorado.edu/pub/SerialEM/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">UCSF-Chimera</td>
			<td style="width:205px;">free download</td>
			<td style="width:119px;"></td>
			<td style="width:447px;"><a href="http://www.cgl.ucsf.edu/chimera/download.html">http://www.cgl.ucsf.edu/chimera/download.html</a></td>
		</tr>
	</tbody>
</table>

helical organization of blood coagulation factor viii on lipid nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

Recombinant human and porcine FVIII were successfully organized helically on negatively charged single bilayer LNT, resembling the activated platelet surface. The helical organization of the human and porcine FVIII-LNT was consistent through the collected digital micrographs (Figure&#160;2). &#160;The control LNT and the human and porcine FVIII-LNT helical tubes were selected and segmented with the e2helixboxer.py GUI and initial data sets created with the e2workflow.py GUI, Single parti...

Watch this Scientific Journal Video about Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes at JoVE.com

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

helical-organization-blood-coagulation-factor-viii-on-lipid

Research

JoVE Journal

Bioengineering

12.2K 조회수.  University of Texas Medical Branch.  인간과 돼지 : 우리는 두 개의 매우 상동 FVIII 형태의 막 결합 구조를 해결하기 위해 적용 극저온 전자 현미경, 지질 나노 기술 및 구조 분석의 조합을 제시한다. 나선형으로 마이너스로 충전 된 지질 나노 튜브 (LNT)에있는 두 개의 기능 재조합 FVIII의 형태를 구성하는 우리의 실험실에서 개발 된 방법론을 설명한다. 