This document summarizes work done to isolate and characterize the cytoplasmic domains of platelet inhibitory receptor proteins. The cytoplasmic domains of G6bB and TLT-1 were amplified by PCR and cloned into expression vectors. Restriction digests and gel electrophoresis confirmed successful cloning. Future work will involve ligating the domains into an NMR expression vector, expressing the proteins, and using NMR to study interactions with lipid membranes. This will provide insight into how these receptors negatively regulate platelet activation.

1. Ibrahim Vazirabad
August 23rd, 2011

2. Introduction to the Role of Platelets in Hemostasis
•Eventually, vessel anucleate blood bind that injury.
•If the blood a thrombus will form injured,to various through the of the matrix.
•Platelets are small wall becomesat the site of travelconstituents bloodstream.
have many receptors that cells extra-cellular matrix becomes exposed.
As the platelets pass, they will encounter ECM and adhere to it through their
various receptors that recognize matrix proteins.
ECM

3. Proposed Model for Initiation of PECAM-1-Mediated Inhibitory Signaling in
Response to GPVI Engagement
1 S
S
PECAM-1
•One such part of the extra-cellular to
•The cytoplasmic domain of PECAM-1
•A receptor on platelets, GPVI, bindsmatrix
2 S
S
Collagen collagen. GPVIITIMs (ImmunoreceptorFcRγ
is collagen.
contains two is associated with the
3 S
S Tyrosine Inhibitory Motifs) which when
chain, which has an ITAM (immunoreceptor
4 S
S
tyrosine-based activation phosphatases which
phosphorylated, recruit motif), which
positively platelet activation. activation and
oppose regulates platelet
GPVI S
S 5 S
S
thrombus formation. Journal of Biological Chemistry.
Mori J.,et al., The
2008; 283: 35419-35427
S
S
FcRg 6 S
S
DAG
SH3
S-S
IP3 Y663
Y686
Syk
Fyn/Lyn
Ca2+
Platelet SHP-2
Activation

4. PECAM-1 of also known to have a phosphorylate Ysegment which allows with
S707 is constitutively phosphorylated in resting platelets,whichwhen S702 is
Activation is a tyrosine kinase will lipid-associated 686, and then interacts
the plasma membrane. .
phosphorylated, of YPECAM-1 cytoplasmic domain begins to dissociate from
phosphorylation the 663
the plasma membrane. Paddock, C.P.,et al., Blood. 2011; 117: 6012-6023

5. Other ITIM-containing Molecules on Platelets
1 S
S
PECAM-1
2 S
S
CEACAM-1
Collagen
3 S
S
1 S
S
4 S
S
2 S
S
GPVI S
5 S G6bB TLT-1
S S
3 S
S
S
S
FcRg 6 S
S
4 S
S 1 S
S 1 S
S
SH3 DAG
S-S
Y663
Y493 Y211 Y246
IP3 Y686
Y237
Y520 Y282
Syk There are other ITIM-containing molecules α-
TLT-1(TREM-like Transcript-1) antigen in
G6b-B also negatively regulates found cell
CEACAM-1(Carcinoembryonicis platelet the
Fyn/Lyn expressed through theis a their suspected the
activationmolecule-1)and are progenitor,
adhesionofon platelets thatIg Fc receptor γ
granules platelets GPVI superfamily
to associate withWhennegativelybecome
glycoproteinalso the plasma membrane in
megakaryocyte. also platelets regulates
chain, but it thatinteracts with the C-type
Ca2+ an analogous waythroughto the platelet
platelet signaling to PECAM-1:
activated, TLT-1 migrates the
lectin-like receptor 2 (CLEC-2).GPVI Fc
CEACAM-1
surface to promote platelet Chemistry. 2008; 283:
receptor Mori J.,et al., The Journal of Biological aggregation.
γ chain.
G6bB
35419-35427
Morales, Wong, C., et al., Blood. 2009; 113: 1818-1828
J., et al., Blood Coagulation & Fibrinolysis. 2010;21: 229-236
Platelet TLT-1
Activation

6. Evidence for PECAM-like Cytoplasmic Behavior in
other ITIM molecules
 Immediately after the PECAM C-terminal ITIM are two positively charged
residues that are hypothesized to allow the cytoplasmic domain to attach to the
plasma membrane.
 Therefore, existence of these basic residues in the cytoplasmic domain of the
other three ITIM-containing molecules is evidence of similar plasma membrane
interaction.
+ residues ITIM/S-T Other residues
ID Sequence pI ++
PECAM-1C TVYSEVRKAVPDAVESRYSR 9.1 Yes
PECAM-1N VQYTEVQVSSAESHKDLGKK 7.5 Yes
CEACAM-1C IIYSEVKKQ 9.3 Yes
CEACAM-1N VTYSTLNFEAQQPTQPTSAS 3.9 No
G6bB-C TIYAVVV 5.2 No
G6bB-N LLYADLDHLALSRPRRLSTA 9.3 Yes
TLT1-C VTYATVIFPGGNKGGGTSCG 8.5 No
TLT1-N TTYTSLPLDSPSGKPSLPAP 6.3 No
CEACAM and G6bB appear to interact with membrane. TLT-1 does not.

7. Do CEACAM,TLT-1, and G6b-B associate with the
membrane in an analogous way to PECAM-1?
 Based on sequencing data, we hypothesize that both G6bB and
CEACAM-1 associate with the plasma membrane. TLT-1 will not.
General Project Steps:
 First, the cytoplasmic domains of the three ITIM-containing molecules
will be cloned into vectors.
 Then, NMR will be performed on the cytoplasmic domains:
1. In aqueous solution
2. With lipid micelles
 This will allow understanding whether membrane interaction takes
place.

8. Step 1:Preliminary Work
 First two proteins under study: G6bB and TLT-1.
 We received plasmids from:
1. G6b-B plasmid: Dr. Yotis Senis
2. TLT-1 plasmid: Dr. Valance Washington
 Dr. Senis sequenced plasmid before sending it, Dr. Washington did
not, so TLT-1 plasmid was sequenced. No surprises.
 We then transformed E. Coli with the two plasmids and harvested DNA
with the QIAGEN Miniprep Kit.

9. Step 2: Isolating Cytoplasmic Domains and PCR
 Primers were designed that would amplify the cytoplasmic domain portion of
the two proteins. RE sites added to allow digest in any vector.
G6bB:
Forward: 5’-ATT GGA TCC TGG CTG CAC AGG CGC CTG CCC-3’
Reverse: 5’-CCC AGG CTT TCA AAC TAC AAC TGC ATA GAT-3’
TLT-1:
Forward: 5’- ATT GGA TCC ATG GCC AAG AAG AAA CAA GGG-3’
Reverse: 5’-CCC AGG CTT GCT GGA TGG AGT CTG ATT GTT-3’
Red- BamHI site Green-HindIII site
 After primers were created, the cytoplasmic domains were amplified via PCR.
The product was then ran on a gel.

10. PCR Product Gel and Extraction
100 bp ladder
1kb ladder
G6Bbcyto
TLT-1cyto
Expected Sizes of Fragments
TLT-1cyto : 390 bp
G6b-Bcyto : 234 bp
Success.
TLT-1 and G6BB bands were then
removed from the gel according to
the QIAGEN gel extraction kit.

11. Step 3: Ligation of PCR product
 The extracted PCR products were then
ligated into a new plasmid, called pCR-Blunt
II-TOPO.
 Used as it is very easy to insert products into
and digest with RE.
 The plasmids were then transformed into E.
Coli, plated on agar.
 Ten colonies picked. (6 G6bB, 4 TLT-1)

12. Step 4: Quality Control
 The TOPO vector has EcoRI restriction sites on either side of the product insert.
 Allows for an easy confirmation of successful ligation.
 All ten clones were digested by EcoRI.
Reaction Mix:
2 ul of NEB buffer 4
400 ng plasmid
0.5 ul of EcoRI HF (20U/ul)
x ul of water (20 ul total)
------------------------------------------------------------------1.5 hr digestion, 37C
 Gel was loaded to visualize digest.

13. EcoRI Digestion Gel
1kb ladder TLT-1 #1-4 G6Bb #1-6
Expected Sizes of Fragments
TLT-1cyto : 408 bp 500 bp
uncut plasmids
G6BBcyto : 252 bp
•No 100 bp ladder, mistake.
•The fragments “look” correct however.
•Next step is to cut with the BamHI and HindIII
sites to ligate into another vector

14. Step 5: BamHI and HindIII Double Digestion
 One G6bB, one TLT-1 clone were double digested by BamHI and
HindIII, along with final plasmid, called pQE30 GB1.
Reaction Mix:
10 ul of NEB buffer 2
7 ug plasmid DNA
2 ul of HindIII (20U/ul)
2 ul of BamHI (20U/ul)
x ul of water (100 ul total)
3 hr digestion, 37C
His Tag: purification, Ni column.
GB-1 Domain: Known chem. shift in NMR
 Agarose gel was loaded TEV: protease which removes first two.
to visualize the double digest.

15. BamHI-HindIII Double Digest Gel
PQE30(uncut)
100 bp ladder
G6Bb(uncut)
TLT-1(uncut)
1kb ladder
G6Bbcyto
TLT-1cyto
PQE30
Expected Sizes of Fragments
TLT-1cyto : 390 bp, 18 bp, 45 bp
G6BBcyto : 234 bp, 18 bp, 45 bp 3 kb
2 kb
1.5 kb
PQE30 plasmid: 15 bp 1 kb
500 bp
•The double digest was 200 bp
unsuccessful. New England Bioscience
discouraged a double digest, so not surprising.
•Ordered the High Fidelity versions of BamHI and HindIII,
which are guaranteed to work together.

16. Troubleshooting
To see whether the BamHI or HindIII  We were unsure of the EcoRI digestion, so
enzymes were inactive, this gel was run. it was repeated, this time with a 100bp ladder.
•Both REs are active.
Success.
•Correct bands.
Success.

17. BamHI-HindIII Double Digest Gel: Repeated
100 bp ladder
PQE30(uncut)
G6Bb(uncut)
TLT-1(uncut)
1kb ladder
G6Bbcyto
TLT-1cyto
PQE30
Expected Sizes of Fragments
TLT-1cyto : 390 bp, 18 bp, 45 bp
G6BBcyto : 234 bp, 18 bp, 45 bp
1000 bp
PQE30 plasmid: 15 bp
500 bp
•Perfect. A gel with large wells will
be loaded to maximize product 200 bp
separated.

18. Large Well Double Digest Gel
uncut G6bB plasmid
uncut TLT-1 plasmid
G6Bb double digest
TLT-1 double digest
Digest Reaction:
100 bp ladder
100 bp ladder
10 ul of NEB buffer 2
5 ug plasmid DNA
2 ul of HindIII (20U/ul)
2 ul of BamHI (20U/ul)
x ul of water (100 ul total)
---------------------------------------3 hr digestion, 37C
•G6Bb band did not show up. 500 bp
•G6Bb band runs at ~250 bp, a dark band can be 300 bp
seen at ~250 bp (marked in red). This is loading dye.
uncut TLT-1 plasmid
TLT-1 double digest
100 bp ladder

19. Future Plans
1. Ligate G6bB and TLT-1 into PQE30 vector.
2. Cut out to ensure successful ligation.
3. Sequence to make sure no mutations.
 CEACAM plasmid has just been sequenced.
 Repeat process.

20. Appendix: PCR Protocol
5 ul of 2ng/ul DNA
5 ul of forward and reverse primers(5 um solution of each)
1 ul of dNTPs(100 mM, 25 mM of each dNTP)
10 ul 10x PCR buffer
73 ul H2O
0.5 ul PFU Turbo (High Fidelity DNA polymerase)
PCR program:
94 C 5’
65°C 3’
72°C 3’
94°C 1’ Bold repeated for 30 cycles
65°C 3’
72°C 7’
10°C ∞

21. Appendix: Ligation Reaction
4 ul of DNA product
1 ul of provided salt solution
1 ul of PCRII Blunt TOPO plasmid vector
Incubate mixture for 5 minutes at room temperature.
Then transform plasmid into OneShot Chemically Competent cells.

22. Appendix: Transformation Protocol
 Aliquot 50 ul of competent E.coli, thaw on ice.
 Add 10 ng of plasmid, let sit for 30 minutes on ice.
 Heat shock mixture for 30 seconds, then let sit on ice for two minutes.
 Add 950 ul of LB broth to the mixture. Incubate on shaker at 250 rpm for
one hour at 37 C.
 Add 100ug/ml ampicillin to the mixture, let incubate for 1 hour.
 Add to 250 ml flask of LB-amp broth.

23. Appendix: Sequencing Protocol
Mixture:
0.5 ug of plasmid DNA
2ul of primer stock at 1.6 uM
X ul of dH2O
2 ul of 5x sequencing buffer
4 ul of Big Dye Terminator Kit
20 ul total volume
Thermocycler Protocol:
96°C 30 sec
50°C 15 sec
60°C 4 min
25 cycles
4°C ∞