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.
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.
Here are a few sequences to get started with.
We recommend searching the databases for more.
- edlslvgqpe ndydtgddbt aadpdsnnta aaldvr
- edgsdppsgd fltegggvr
- aevqdkgefl aegggvr
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/ )
- Compare trees generated with the fossil record or the tree of life.
- Compare rate of change in fibrinopeptides with other protein families.
- 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.