Fibrinopeptide Laboratory

Background

Scientists have always been fascinated by how blood clots. Early efforts were directed at dissecting the vertebrate coagulation system and determining its components. This work revealed the central event in blood coagulation to be the conversion of soluble fibrinogen to insoluble fibrin. Basically, this is accomplished when the coagulation enzyme thrombin (factor IIa) removes small polar peptides (termed the fibrinopeptides) from each fibrinogen molecule forming fibrin. These fibrin molecules non-covalently interact with each other forming a fibrin web. Fibrin stabilization is accomplished by the action of a second coagulation enzyme (factor XIIIa) that introduces numerous covalent crosslinks between these fibrin molecules. The resulting fibrin web is able to capture platelets and red blood cells, effectively sealing the wound and stemming plasma (fluid) loss. Fibrinogen (also called factor I) is a postranslationally modified plasma protein composed of three pairs of polypeptide chains (Aa₂ Bb₂g₂), with an

average molecular weight of 340,000 daltons. Between 1.7 and 5.0 grams of fibrinogen are synthesized per day by the liver with approximately 75% of this fibrinogen excreted into the plasma. This translates into a mean plasma level of 3-4 mg/mL with a normal half-life of 3 to 5 days. Many early investigators tried to determine the differences between fibrinogen and fibrin. Their molecular weights were identical and fibrinogen was the more electronegative of the two. In the conversion of fibrinogen to fibrin, thrombin catalyzes the hydrolysis of Arg-Gly bonds removing small, polar amino terminal pieces of what was later determined to be both the a and b chains. Termed fibrinopeptides A and B (FPA and FPB respectively), they varied in length between 13 to 21 amino acids in various mammals (thus totaling less than 2% of the total mass) with a total charge of between -2 to -6. Bettelheim and Bailey first hypothesized that these newly exposed amino termini ("knobs") must be the principle contact sites during polymerization. In human fibrinogen, the a chain "knob" begins with the sequence GPRVV while the b chain starts with GHRPL. Synthetic GPR derivatives based on the a "knob" were found to bind to fibrinogen, and inhibit fibrin monomer polymerization. Peptides based on the b "knob" also bound to fibrinogen, but were unable to inhibit fibrin monomer polymerization. In analogous studies, venoms can be used to selectively remove FPA without activating factor XIII and fibrin will still form. Although it appears morphologically the same, it lacks the normal strength of fibrin. Removal of only FPB (without FPA release) results in fibrin formation in lampreys but human fibrinogen clots only if the temperature is maintained below 15C. In fact, Doolittle et al. postulated that the b "knobs" act like claspers in b "holes" thus holding abutting molecules together rather than being involved in lateral growth. Early phylogenetic work on fibrinogen began with protein sequences obtained for various fibrinopeptides. It was immediately obvious that although these peptides were exceptionally variable, though certain features were conserved; without exception they all contained an arginyl-glycine bond required for thrombin cleavage.

Molecular Clocks

The longer two species have been evolving separately, the more amino acid differences accumulate in their proteins. Amino acid changes reflect mutations in genes. The basic mutation rate is probably similar for all genes, but natural selection filters out those mutations that impair a protein's function. Molecular clocks "tick" at different rates, and the same protein may change somewhat faster in one lineage, like rodents, than in another, like primates. To get the best time estimates of divergence between species, it is necessary to check the rate of molecular change against events in the fossil record, if there is one. Molecular clocks are not precise like digital clocks, so it is a good idea to use several different molecules if possible.

Data

Here are a few sequences to get started with.


1. edlslvgqpe ndydtgddbt aadpdsnnta aaldvr
2. edgsdppsgd fltegggvr
3. aevqdkgefl aegggvr
4. nqeglfhgr
We recommend searching the databases for more.

Tools

Biology Workbench (http://workbench.sdsc.edu)

The Tree of Life (http://tolweb.org/tree/phylogeny.html)

SwissPDB Viewer (locally installed-- but get your own copy here http://us.expasy.org/spdbv/ )

Potential Investigations

• Compare trees generated with the fossil record or the tree of life.
• Compare rate of change in fibrinopeptides with other protein families.

Goals

• Find the data & use the tools provided to explore these questions.
• Share your experiments and insights with the group.
• Evaluate the effectiveness of this exercise and these two sessions.