Summary
To evaluate the role of hydrophobic bonding in coagulation reactions, evidence was
collected from the literature and amplified with studies of clotting factors adsorbed
onto hydrophobic and hydrophilic surfaces.
Resin thrombin, Thrombin Topical, and human factor V were found to lose their clotting
activity when adsorbed onto excessive amounts of barium stearate or aluminium octoate,
while bovine factor V remained active. Adsorbed onto a limited area, thrombin remained
active and may have been deposited in multiple layers.
Presence and polarity of residues left by serum, plasma, albumin, thrombin, fibrinogen,
and fibrinogen overlaying thrombin, upon glass and plexiglas plates, could be demonstrated
by exposure of the adsorbates to oxides of certain transition elements and to condensing
water vapor.
A recording ellipsometer was used to measure amount and rate of adsorption at a solid/liquid
interface, for thrombin and for fibrinogen and plasma constituents upon thrombin,
using the following optically sensitive hydrophilic and hydrophobic surfaces: anodized
tantalum sputtered glass slides, with or without a coating of barium lignocerate (TaN
and TaW slides respectively), and polished slices of silicon crystals cleaned with
hydrofluoric acid, with and without a final exposure to boiling nitric acid (SiW and
SiN slides respectively). Calibration of machine and surface properties is described.
The source of thrombin preparation rather than the wettability of the solid appeared
to affect the adsorption curve; about 25 A. of Resin thrombin was adsorbed linearly
on TaW and TaN slides in two minutes, after which the adsorption rate decreased to
near zero, while the initial adsorption rates of Thrombin Topical were greater and
decreased slowly until 30 to 60 A. were adsorbed in 20 minutes. Slides could be dipped
before, and also after the protein had been added to the solution, allowing repeated
adsorption curves to be made. Only TaN surfaces could thus be shown to pull down a
preformed film of thrombin adhering to the slide at the air/liquid/solid interface;
more thrombin was then adsorbed from the solution to complete the process. Deposited
on any of the four surfaces used, thrombin could adsorb 30 to 55 A. of fibrinogen
out of fraction I solutions and cause its polymerization. The resulting fibrin formation
on the slide and, frequently, in the solution, caused oscillations and an increased
overall light output as shown by the recorder, but did not affect the rate of fibrinogen
adsorption calculated from relative amplitude and phase shift of transmitted light.
Addition of monochloroacetic acid abolished the oscillations and caused a decrease
of apparent recorded and calculated thickness. From dilute ACD plasma, Resin thrombin
on TaN slides adsorbed only approximately 20 A. of what we presume to be fibrinogen,
while on the other surfaces 40 to 60 A. was adsorbed.
Combining literature and experimental data, we propose two examples of models which
allow a role for hydrophobic bonding in the following reactions:
1. After contact with glass, hydrophobic bonds in factor XII are exposed, and combine
with those available on factor XI;
2. After an electrostatic reaction in which positive charges on product I are aligned
to pull several phospholipid molecules out of a micelle, the apolar fatty acid tails
thus exposed adsorb factor V at its hydrophobic bond site;
3. When prothrombin is converted to thrombin, hydrophobic sites are exposed which
will be adsorbed at similar sites on fibrinogen, thus aiding the alignment of the
enzyme.
In general, coagulation may then be seen as the initiation and transfer of exposing
hydrophobic bonds to the aqueous phase.
