Ibrahim Vazirabad  August 23rd, 2011
Introduction to the Role of Platelets in Hemostasis•Eventually, vessel anucleate blood bind that injury.•If the blood a th...
Proposed Model for Initiation of PECAM-1-Mediated Inhibitory Signaling in                       Response to GPVI Engagemen...
PECAM-1 of also known to have a phosphorylate Ysegment which allows with S707 is constitutively phosphorylated in resting ...
Other ITIM-containing Molecules on Platelets                                          1    S                              ...
Evidence for PECAM-like Cytoplasmic Behavior in                other ITIM molecules Immediately after the PECAM C-termina...
Do CEACAM,TLT-1, and G6b-B associate with the membrane in an analogous way to PECAM-1?     Based on sequencing data, we h...
Step 1:Preliminary Work   First two proteins under study: G6bB and TLT-1.   We received plasmids from:    1. G6b-B plasm...
Step 2: Isolating Cytoplasmic Domains and PCR Primers were designed that would amplify the cytoplasmic domain portion of ...
PCR Product Gel and Extraction                                    100 bp ladder                                           ...
Step 3: Ligation of PCR product The extracted PCR products were then  ligated into a new plasmid, called pCR-Blunt  II-TO...
Step 4: Quality Control       The TOPO vector has EcoRI restriction sites on either side of the product insert.       Al...
EcoRI Digestion Gel                                          1kb ladder   TLT-1 #1-4   G6Bb #1-6Expected Sizes of Fragment...
Step 5: BamHI and HindIII Double Digestion One G6bB, one TLT-1 clone were double digested by BamHI and  HindIII, along wi...
BamHI-HindIII Double Digest Gel                                                   PQE30(uncut)                            ...
TroubleshootingTo see whether the BamHI or HindIII        We were unsure of the EcoRI digestion, soenzymes were inactive...
BamHI-HindIII Double Digest Gel: Repeated                                                                                 ...
Large Well Double Digest Gel                                                                                              ...
Future Plans1.   Ligate G6bB and TLT-1 into PQE30 vector.2.   Cut out to ensure successful ligation.3.   Sequence to make ...
Appendix: PCR Protocol5 ul of 2ng/ul DNA5 ul of forward and reverse primers(5 um solution of each)1 ul of dNTPs(100 mM, 25...
Appendix: Ligation Reaction4 ul of DNA product1 ul of provided salt solution1 ul of PCRII Blunt TOPO plasmid vectorIncubat...
Appendix: Transformation Protocol Aliquot 50 ul of competent E.coli, thaw on ice. Add 10 ng of plasmid, let sit for 30 m...
Appendix: Sequencing ProtocolMixture:0.5 ug of plasmid DNA2ul of primer stock at 1.6 uMX ul of dH2O2 ul of 5x sequencing b...
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BRI Summer 2011 PECAM Project

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This presentation was the culmination of my research at the Blood Research Institute of Wisconsin.

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  • Blood provides essential nutrients heart moves blood, creates a pressurized systems in arteries clotting factors
  • Collagen binding to GPVI enables the SFKs, Lyn and Fyn, to simultaneously phosphorylate FcRγ and Y686 of PECAM-1, priming PECAM-1 for phosphoryla-tion on Y663. Syk then binds phosphorylated FcRγ and phosphorylates LAT, creating binding sites for the SH2 domain-containing proteins, Gads/SLP-76, PI3K, and PLCγ2. PI3K converts phosphatidylinositol (4,5) bisphosphate (PIP2) to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and Syk phosphorylates SLP-76, allowing recruitment of PLCγ2 and Btk via their pleckstrin homology and SH2 domains, respectively. Btk phosphorylates and activates PLCγ2 to cleave PIP2 to produce IP3 and DAG, culminating in Ca2+ releaseand platelet activation. Btk associates with pY686 of PECAM-1 and phosphorylates Y663. The dually phosphorylated ITIMs of PECAM-1 bind SHP-2 and inhibit GPVI signaling.
  • Collagen binding to GPVI enables the SFKs, Lyn and Fyn, to simultaneously phosphorylate FcRγ and Y686 of PECAM-1, priming PECAM-1 for phosphoryla-tion on Y663. Syk then binds phosphorylated FcRγ and phosphorylates LAT, creating binding sites for the SH2 domain-containing proteins, Gads/SLP-76, PI3K, and PLCγ2. PI3K converts phosphatidylinositol (4,5) bisphosphate (PIP2) to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and Syk phosphorylates SLP-76, allowing recruitment of PLCγ2 and Btk via their pleckstrin homology and SH2 domains, respectively. Btk phosphorylates and activates PLCγ2 to cleave PIP2 to produce IP3 and DAG, culminating in Ca2+ releaseand platelet activation. Btk associates with pY686 of PECAM-1 and phosphorylates Y663. The dually phosphorylated ITIMs of PECAM-1 bind SHP-2 and inhibit GPVI signaling.
  • BRI Summer 2011 PECAM Project

    1. 1. Ibrahim Vazirabad August 23rd, 2011
    2. 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 theirvarious receptors that recognize matrix proteins. ECM
    3. 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 SykFyn/Lyn Ca2+ Platelet SHP-2 Activation
    4. 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 interactsthe 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. 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 theFyn/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. 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 residuesID Sequence pI ++PECAM-1C TVYSEVRKAVPDAVESRYSR 9.1 YesPECAM-1N VQYTEVQVSSAESHKDLGKK 7.5 YesCEACAM-1C IIYSEVKKQ 9.3 YesCEACAM-1N VTYSTLNFEAQQPTQPTSAS 3.9 NoG6bB-C TIYAVVV 5.2 NoG6bB-N LLYADLDHLALSRPRRLSTA 9.3 YesTLT1-C VTYATVIFPGGNKGGGTSCG 8.5 NoTLT1-N TTYTSLPLDSPSGKPSLPAP 6.3 No CEACAM and G6bB appear to interact with membrane. TLT-1 does not.
    7. 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. 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. 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. 10. PCR Product Gel and Extraction 100 bp ladder 1kb ladder G6Bbcyto TLT-1cytoExpected Sizes of Fragments TLT-1cyto : 390 bp G6b-Bcyto : 234 bpSuccess.TLT-1 and G6BB bands were thenremoved from the gel according tothe QIAGEN gel extraction kit.
    11. 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. 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. 13. EcoRI Digestion Gel 1kb ladder TLT-1 #1-4 G6Bb #1-6Expected 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 HindIIIsites to ligate into another vector
    14. 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. 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 Biosciencediscouraged a double digest, so not surprising.•Ordered the High Fidelity versions of BamHI and HindIII,which are guaranteed to work together.
    16. 16. TroubleshootingTo see whether the BamHI or HindIII  We were unsure of the EcoRI digestion, soenzymes were inactive, this gel was run. it was repeated, this time with a 100bp ladder. •Both REs are active. Success. •Correct bands. Success.
    17. 17. BamHI-HindIII Double Digest Gel: Repeated 100 bp ladder PQE30(uncut) G6Bb(uncut) TLT-1(uncut) 1kb ladder G6Bbcyto TLT-1cyto PQE30Expected Sizes of FragmentsTLT-1cyto : 390 bp, 18 bp, 45 bpG6BBcyto : 234 bp, 18 bp, 45 bp 1000 bpPQE30 plasmid: 15 bp 500 bp•Perfect. A gel with large wells will be loaded to maximize product 200 bpseparated.
    18. 18. Large Well Double Digest Gel uncut G6bB plasmid uncut TLT-1 plasmid G6Bb double digest TLT-1 double digestDigest 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 bpseen at ~250 bp (marked in red). This is loading dye. uncut TLT-1 plasmid TLT-1 double digest 100 bp ladder
    19. 19. Future Plans1. 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. 20. Appendix: PCR Protocol5 ul of 2ng/ul DNA5 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 buffer73 ul H2O0.5 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 cycles65°C 3’72°C 7’10°C ∞
    21. 21. Appendix: Ligation Reaction4 ul of DNA product1 ul of provided salt solution1 ul of PCRII Blunt TOPO plasmid vectorIncubate mixture for 5 minutes at room temperature.Then transform plasmid into OneShot Chemically Competent cells.
    22. 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. 23. Appendix: Sequencing ProtocolMixture:0.5 ug of plasmid DNA2ul of primer stock at 1.6 uMX ul of dH2O2 ul of 5x sequencing buffer4 ul of Big Dye Terminator Kit20 ul total volumeThermocycler Protocol:96°C 30 sec50°C 15 sec60°C 4 min25 cycles4°C ∞

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