00:01
Oh boy, now we're back to coagulation factors.
00:05
And remember, the intrinsic pathway,
that's driven off a negatively charged surface.
00:10
Remember, the extrinsic pathway,
that starts with tissue factor,
otherwise known as thromboplastin or as factor III
and then we get to the final common pathway
and we activate prothrombin it becomes thrombin,
we cleave fibrinogen to become fibrin,
we crosslink it with XIII.
00:26
Yeah, you now know that in your sleep
and probably wakes you up with nightmares.
00:31
Okay, but now we need to
measure various aspects of this
and another reason that we have
conveniently divided things
into an intrinsic pathway
and an extrinsic pathway,
is because, our different clotting
parameters, our different tests,
will look at those somewhat independently.
00:49
So, measuring coagulation.
00:52
The, PTT, remember we talked about the PT and PTT.
00:55
Partial thromboplastin time, will look
primarily at the intrinsic pathway.
01:00
It clearly also involves components
of the final common pathway,
just by virtue of the way we do the test
and we'll talk about that momentarily.
01:08
So that's the PTT, the intrinsic pathway.
01:12
And then we have the prothrombin time or PT,
which looks at primarily, the extrinsic pathway
and again, there's overlap, both of these times,
both of these clotting parameters
that we can measure in the laboratory.
01:27
We'll also look at prothrombin activity
and fibrinogen cleaving et cetera.
01:33
Okay, let's look at these
in a little bit more detail.
01:37
We'll start first, with a prothrombin
time and again prothrombin time,
that's going to be looking at
mostly the extrinsic pathway.
01:45
So, when we first described this back in 1935,
only prothrombin or factor II was known,
so that's why it has got
that name as prothrombin time
and basically, we would take the patient's plasma,
notably, take a step back, in all
of these coagulation measurements,
we have to draw the patient's blood in such a way,
that we prevent it from clotting in the test tube.
02:10
If I simply take your blood,
suck it up into a test tube,
leave it on the shelf, it will clot.
02:15
The coagulation factors will
get stochastically activated
and you're off and running and over
a period of time they will clot.
02:23
So, we inhibit the clotting
when we draw the blood,
by putting in a chelator of calcium,
ethylenediaminetetraacetic,
EDTA, is commonly used.
02:35
Citrate is commonly used and what that does is,
it sucks up all the calcium,
that's in the blood and prevents
the from clotting in the
test tube until we're ready.
02:44
Okay, so, we take the patient's
blood and it’s not clotted
because we've added that chelator,
we spin down all the red cells,
we spin down all the platelets
and all we have left is the
protein, the plasma on top.
02:56
We take that into a separate
tube or tray, we add back calcium
and then we add the start component,
in this case, because it's the extrinsic pathway,
we're giving back thromboplastin,
we're giving back tissue factor, factor III.
03:14
And what is that?
So, it's a tissue extract,
originally it was from rabbit brains,
interestingly enough,
but it contains a lot of phospholipid
and it contains a lot of tissue factor.
03:23
Okay and so we take the plasma,
we add back calcium, we give that thromboplastin
and we start our stopwatch and then we
measure how long it takes to get a clot
and it all begins with that factor VII activation,
interacting because of the
thromboplastin or the tissue factor.
03:42
And it measures, factors III, that's
the thromboplastin or tissue factor,
factor VII, X, V, II, and I.
03:50
And it gets it gives us a time it's
usually in the ballpark of 12 seconds
or thereabouts and we express it,
as an international normalization ratio or “INR.”
So, in other words, we will
take a completely normal person
and we'll see how long it takes to
get clotting through this pathway
and then we'll take the
unknown sample from a patient
and we'll see how long it takes
and then we'll compare that
as a ratio and normally 1,
is where you want to be, because
that means you're normal,
if it's longer than one,
then you have abnormal clotting and sometimes,
that's because, we've administered
therapy to make it abnormal
and sometimes it's because
you have other problems.
04:34
We typically use, the INR
from the prothrombin time,
to assess coumadin therapy and
coumadin is a factor K antagonist,
that we'll talk about
subsequently in another session,
that blocks the synthesis of
factors II, VII, IX, and X.
04:54
So, important in this. Okay.
04:58
So that's prothrombin time.
05:00
Let's look on the other side,
how are we going to measure the
intrinsic pathway components?
and this is the partial
thromboplastin time or PTT.
05:09
Okay, so when this was first described,
this is mid 40s.
05:14
Partial thromboplastin had phospholipid in it,
but didn't have tissue factors,
so it wasn't called, it wasn't the
same test as the prothrombin time,
which looked at tissue factor
activation of the extrinsic pathway.
05:27
Again, what are we doing?
So, we take the patient plasma again,
it's anticoagulated, so we have we
have chelated up all the calcium
and now we add back calcium
and we add back phospholipid
and normally the phospholipid
in the normal scheme of things,
would be provided by the platelet plasma membrane
or by the polyphosphates
being secreted by platelets
and then we add an intrinsic pathway activator.
05:56
And in most laboratories,
that's just a glass bead,
it's a surface upon which the things can occur,
so that's how we actually do the test
and the intrinsic pathway
measures the time to form a clot
after that factor XII activation,
factor XII is being activated by those glass beads
and then in the presence of the
phospholipid and the calcium,
we go through the intrinsic
pathway and get a clot.
06:22
So, we want to convert fibrinogen into fibrin
and what we're measuring here is,
factors: XII, XI, IX, VIII, X, V, II, and I.
06:31
And we commonly use this to assess
the efficacy of heparin therapy,
remember in the previous session,
we talked about giving heparin
because it interacts with antithrombin III,
which blocks, some of the
components of the intrinsic pathway.
06:48
To see how well we're doing
with our heparin therapy,
we can do a partial
thromboplastin time or a PTT test.
06:56
Okay, so we have the extrinsic pathway through PT,
we have the intrinsic pathway through
the partial thromboplastin time,
we use the PT to assess the
efficacy of coumadin therapy,
we use partial thromboplastin time, PTT,
to assess the efficacy of heparin.
07:14
And with that, you've gotten the gist
of how we measure coagulation factors.