Charles Kreisel


 


 Journal/Papers  Review/Comments
January 30 - NIH Public Access: Thrombin 

I began to read the first few pages of  ThrombinPaper.pdf and I learned some things about thrombin and its various biological functions.

I also learned about some basic ligands which bind to thrombin and what their functions are. It seems to me that the sodium ion is of particular importance.

I'll be on the lookout for thrombin structures in the PDB with sodium binded to them and I'll save them in my personal annotations. I plan to read more of this paper tomorrow;

it seems to me that it will be helpful.

February 1 - NIH Public Access: Thrombin

 I continued to read further into ThrombinPaper.pdf which delved deeper into the purposes of sodium ions in the cleavage of many PAR's

(Protease Activated Receptors) as well as the binding of thrombomodulin to thrombin as a surpesor of the ability of thrombin to coagulate,

and which in turn increases specificity towards zymogen protein c, leading to anti-coagulation. Some other noteworthy processes include the binding of sodium

ions as a necessity for the optimal cleavage of fibrinogen and the necessity for factors V, VI, and XI in the explosive generation of thrombin in the coagulation cascade.

 In total, Na+ essentially determines the procoagulative/anticoagulative fate of thrombin. Following this, I learned more about the structure of thrombin regarding the A chain

and the B chain and the necessity of each for the other. It is noteworthy to mention the signnificant number of negatively charged residues closer to the C-terminus which is

near the positive Na+ site. This appears to guide many interactions. The A-chain is optimally shaped for communication between the Na+ site and the active site of thrombin.

I will finish up the last of this section on thrombin structure tomorrow morning before my meeting. I will also try to extract some more thrombin structures from the PDB and

archive them in my personal annotations section. 

February 2 - PDB Thrombin Search

 This afternoon I searched up several structures of thrombin on the PDB and since Ihave read so much about the power of sodium binding in thrombin, I decided that I

would search for those structures containing the sodium ion. I found many structure hits, many regarding inhibitors, and I will sort through these more later. I saved

roughly 10 structures today and I am going to search some more later, possibly using Activated Protein C and a chemical keyword.

 February 2 - X- Ray Crystallography Paper

This evening I sat down and looked through all of the power points on x - ray crystallography to write my report. 

I also used some other websites to clear up any questions I had. Then when you revised it and sent it back to me, I fixed

the mistakes for my personal reference. 

 February 3 - Molecular View of the Human Anatomy

 Today I attended the lecture on Electron Microscopy, the second experimental method for solving macromolecular 

structure. This lecture was as equally informative as it was confusing at some points, but I will just have to look 

over the power point on my own time to truly understand it. I will email you with any questions I may come across.

I assume that my homework is extended from the previous week and I will email you a 1 page paper on EM by this

coming Thursday. 

 February 4 - NIH Public Access: Thrombin

I finished reading my article of thrombin by Di Cera and it mentioned a few words that I've come accross in my thrombin searches in the PDB. These words 

include PPACK (H-D-Phe-Pro-Arg- CH2Cl) which is an irreversible active site inhibitor, Hirudin which we've seen in several thrombin complexes, and heparin which I've also 

seen in quite a few thrombin complexes. It also stated some more direct locations of Na+ binding as well as Ca2+ binding. I will pick up on reading the other articles on

or Monday. Perhaps some other perspectives can shed some light on other aspects of thrombin and locations of binding sites. 

 February 7 - Direct Thrombin Inhibitors

 I read the article you sent me on DTI's (Direct Thrombin Inhibitors) DTI.pdf This told me a lot about the function of heparin-thrombin complexes.

For example a heparin-thrombin-antithrombin complex involves the binding of heparin to both the exosite II of thrombin and antithrombin simultaneously

thus inhibiting the coagulatory functions of thrombin. However, just as heparin can induce inhibition, it can also for a complex with fibrin in which heparin

acts as a bridge between fibrin and thrombin. Fibrin binds to exosite I on thrombin while heparin binds to fibrin and exosite II. Because both of the exosites

are occupied, antithrombin cannot inhabit the exosite of thrombin and protects coagulation from inactivation. Thus, heparin, when in complex with certain

substrates, can both activate the inhibition of thrombin as a coagulatory protein as well as protect it from inactivation. However, the main point of the

article is to outline several direct inhibitors of thrombin. These inhibitors can be broken down into two sub categories: bivalent (occupying both the active

site and exosite I) and univalent (binding only to the active site). Bivalent DTI's can temporarily inactivate thrombins coagulatory functions, but eventually

are cleaved by thrombin and as a result, only produce a transient inhibition. However, univalent DTI's, by binding only to the active site, can inactivate

fibrin bound thrombin and eventually dissociate from thrombin, leaving a small amount of enzymatically active thrombin available for coagulation.

Another very important property of DTI's is that they act independently of antithrombin. Because of this, DTI's can avoid the problem of the

thrombin-heparin-fibrin complex; they are able to inactivate thrombins coagulatory functions even when heparin is in complex with fibrin and thrombin.

This is important because DTI's can be used clinically in patients who have illnesses such as  thrombocytopenia and people who suffer from

venous thrombosis.  

 February 8 - Thrombin: Structure, Functions, and Regulation

After reading a few sentences of Thrombin Structure_Function_Regulation.pdf, I realized that it is just a condensed, more concise version of 

ThrombinPaper.pdf. This being said, it seemed to include some different information which is very helpful for understanding various structures of thrombin.

There are three forms of thrombin: E, a slow and inactive form of thrombin, E:Na, a fast and active form of thrombin, and E*, an interesting form which is

unable to interact with Na activation. The main differences include the orientation of the D189 the primary specificity pocket and the architecture of the water 

network. In E:Na there is a network of 11 water molecules which connct through H-Bonds. In E there is a network of only 7 water molecules qhich severely 

reduces the conectivity. This network of water molecules is important in communication between the Na site and the S195 active site.  

The paper also discusses the function of several substrates binding to thrombin such as thrombomodulin and Na. Di Cera states that when Na binds to thrombin, it enhances

activity towards procoagulant and prothrombotic substrates such as fibrinogen and protease activates receptors. When thrombomodulin binds to the exosite I, the activity 

towards anticoagulant Protein C is enhanced. Activated Protein C inactivates factors involved in the coagulation cascade. Without thrombomodulin, thrombin has very little 

affinity for Protein C. Is thrombomodulin the same thing as antithrombin? Di Cera uses thrombomodulin, but the authors of DTI's use antithrombin. They appear

to have the same function. Also, the difference between Hirudin and Hirugen is the Hirugen is a peptide derived from the C-terminal fragment Hirudin. 

 February 9 - Thrombin: Structure, Functions, and Regulation  I continued reading about PAR's (Protease Activated Receptors) and the functions of complexes of thrombin with PAR's.
 February 10 - Molecular Visualization Assignment

 Today I tried the molecular visualization assignment assigned to the Molecular View class. I feel much more comfortable with using chimera now and

I am going to try the more advanced assignment later today or tomorrow. 

February 13-14

 Is thrombomodulin an inhibitor? From my readings I have found that they refer to it as a cofactor which increases the specificity towards Protein C

which is an anticoagulant that deactivates the coagulation cascade. Also, in one article by Di Cera, he mentions that binding of Na+ increases specificty

towards prothrombotic and procoagulant substrates. But in another article, he lists the actual substrates which Na+ binding increases

specificity towards and he includes substrates such as thrombomodulin and anti-thrombin III. Thrombomodulin and anti-thrombin III are both anticoagulant

substrates.  

Iam going to make a preliminary list of thrombin inhibitors that I've come across in my readings:

-Antithrombin

-Heparin (in complex with antithrombin)

-Protein C

-Hirudin

 

I am going to do PDB searches for thrombin structures with these inhibitors.  

 February 15

I remember seeing a thrombin structure on the PDB with an oligonucleotide attached to it, and I decided that this might be a point of interest

when making my poster and choosing specific structures of thrombin to display as unique. Luckily I saved this particular structure of thrombin (1HAO) I then decided to

search the title of this PDB entry on PubMed and I found the full length article associated with it. Thrombin Binding Aptamer.pdf Apparently, this oligonucleotide is called

Thrombin Binding Apaptmer.

"In 1992, Block et al. (2) screened a pool of 10 13 synthetic oligonucleotides for their interaction with thrombin, which is

a serine protease with multiple functions in hemostasis whose roles in coronary heart disease and other thrombotic disordershave promoted efforts toward the identification

of specific inhibitors. This study led to the identification of a consensus DNA 15mer, namely GGTTGGTGTGGTTGG (TBA: thrombin binding aptamer), which was found to be a

potent inhibitor of thrombin with an EC50 of 20 nM in a fibrinogen clotting assay (3,4). "

 

According to the authors of this paper, TBA is actually an inhibitor of thrombin, and also appears to be an unusual one. I would like to look more into this as a point of interest.

 February 17

 Today I began trying to create some classification categories for thrombin structures. I visualized a few of the ones I've already pulled from the

PDB and I decided that a decent place to start would be the prescence and locations of the inhibitors/activators.  So far I've categorized a few as having

Exosite I or Exosite II inhibitors. Also, I've classified all of the Na+ bound structures as E:Na+ fast form. I also classified the one unusual E* form of thrombin.

I will spend tomorrow pulling more thrombin structures off of the PDB and perhaps I will continue with classification. One thing I can't seem to figure out

is where is the NAG (N-Acetyl-D-Glucosamine) ligand bound to? It doesn't seem to fit into any of the sites yet it appears in so many structures of thrombin

integrated into the exoskeleton of the chain and attached to an ASN amino acid.

 February 19

 I continued to try and classify some structures of thrombin based on some obvious characteristics including:

Ligand bound or not ligand bound

Site of binding of ligands, inhibitors, etc.

Whether or not the thrombin is being inhibited

E:Na, E, or E* 

 

My main question is what is the role of NAG in thrombin? I did a little research and I think that there is something special about the integration

of NAG binding to an oxygen atom, but I can't find any research on NAG bound to thrombin. 

 February 20  Some more inhibitors I've come across include Boophilin, NAPAP, and MQPA.
 February 22

 I was reading one of the abstracts of a PDB entry when I saw something on the potency of bisphenylic thrombin inhibitors. It

said, "We present a case study of serine protease inhibitors with a bis(phenyl)methanemoiety binding into the S3 pocket. These inhibitors bind with remarkable

 potency to the active site of thrombin, the blood coagulation factor IIa." This bis(phenyl)methane moiety that they're referring to is beta-phenyl-D-phenylalanyl-N-(3-

chlorobenzyl)- L-prolinamide which is a ligand which I've actually seen a lot of, but I never really knew it's purpose. Now I know that it must be related to the

inhibiting of thrombin at the active site. This is something I've yet to see much of. Usually I see inhibitors bound to the exosites. I will start adding the

presence of BPP (not an a professional abbreviation, but I will use it) to my list of classification criteria. Further investigation of BPP reveals that it serously messes up the 

active site and almost seems to mess with the Na+ site as well. 

February 23  Today I wrote uo my first draft of my abstract for the ARC symposium. I sent it to Dr. Dutta.
February 24  I revised my abstract to meet Dr. Dutta's revisions, I wil talk to Dr. Dutta about it one more time tomorrow before I submit it.
February 25

 It seems that many inhibitors of thrombin inhibit at the active site which appears to also mess up the Na+ binding site. I'm not sure if this

is significant but it might be. Also, I'm going to ask Dr. Dutta about retro binding inhibitors today because it seems that there are quite a few of them and they

seem similar to bisphenylic inhibitors, except they don't always have two phenyl groups. 

February 26

 I'm continuing to classify the remainging thrombin structures. Note to self: Na+ Site is between 186 and 225. Use a reference point. Theres

a group of thrombin structures in comples with staphylocoagulase. It is notable to mention this; however, staphylcoagulase doesn't really

inhibit thrombin, it bypasses it. At least I think this is technically not inhibition. 

February 27

 Today I continued to classify more thrombin sturctures. I have 120/365 done and I will continue to classify them the rest of this week. I finally found

structures in complex with antithrombin and heparin. I also noticed some heparin mimetics which inhibit thrombin in the presence of antithrombin. Without

heparin, antithrombin is a poor inhibitor of thrombin. I've seen a lot of structures relating to Factor VIIa and it being inhibited. Why am I finding these

under my thrombin searches? I understand they're pivotal for the coagulation cascade, but they're never in complex with thrombin. 

March 2 I continued classifying the thrombin structures by their ligands and other residues bound to them.
March 3

 I finished classifying the thrombin structures by their ligands and other residues bound to them. Tomorrow morning I will organize these strcutres into separate

lists. 

March 4

 Today I organized my list into categories of ligand and modified residues. This following week I will mostly be looking at separating the phenyl like

inhibitors further. Hopefully I can find a few distinct sub categories for them. 

March 9

 Today I organized my list to make it include only inhibitors. I also added some less common inhibitors to the list. This following week I will further examine

the phenyl like inhibitors and I should hopefully be able to get them done once I have the full remediated files. 

March 21  Today I got the remediated files and I unzipped the folder. I will take the tutorial on making videos later.
March 22

 Today I received the full remediated list of thrombin structures and their types of inhibitors. I fixed up the files a bit by removing non-thrombin entries and

highlighting some structures that may be incomplete. I also took the tutorial on making videos and made a few basic practice videos using thrombin. 

March 31

 Today I classified my list of inhibitors. it seems that the number of amides and ketones are significant to the binding of the inhibitor

to nearby amino acids. Also, note that it seems that non-aromatic rings appear to pull a hydrogen away from the nearby NH2 in the inhibitor

which makes the inhibitor incapable of binding to Gly 216. However, benzene rings and methane seem to leave the NH2 intact. 

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
AttachmentSize
ThrombinPaper.pdf5.22 MB
DTI.pdf308.12 KB
Thrombin Structure_Function_Regulation.pdf290.9 KB
Thrombin Binding Aptamer.pdf2.51 MB
Classifications of Thrombin.xlsx13.14 KB
Thrombin Inhibitors.xlsx14.15 KB
movie1.mp44.54 MB
Thrombin Poster.ppt2.03 MB