Basics of Haemostasis: How to clot, but not too much

I've blogged about the clotting pathway before. However, this lecture focused on different parts of the clotting pathway than my previous post, so yes, here is another post about blood clotting!

Primary haemostasis: Role of platelets and Von Willebrand Factor

Haemostasis is a fancy way of saying "clot formation." There are multiple steps involved in haemostasis:

1. Blood vessels restrict to reduce blood flow to the injured area, thus minimising blood loss.
2. The glycoprotein 1b/IX/V complex on the surface of platelets binds to von Willebrand factor (vWF), which then binds to the subendothelial matrix of the blood vessel (which was exposed by the injury).
3. The exposed collagen on the blood vessel, along with vWF, thrombin, and ADP, work together to activate platelets. Activation of platelets causes them to change shape, express a receptor for a protein called fibrinogen, and release granules containing mediators that help to recruit other platelets.
4. Platelets form an unstable platelet plug.
5. As a result of the clotting cascade, fibrinogen is converted into fibrin, which binds to the fibrinogen receptors (a.k.a. glycoprotein IIb/IIIa) and helps to stabilise the platelet plug.

It is also important to understand why clots don't form when you don't want them to form. Intact endothelium produces NO and prostacyclin, both of which prevent platelets from adhering to healthy endothelium. Furthermore, healthy endothelium shouldn't have exposed subendothelium or collagen for the platelets to bind to.

Clotting cascade: Generation of a fibrin clot

The clotting cascade, which ultimately converts fibrinogen to fibrin, uses a lot of enzymes known as clotting factors. There are two main pathways: the intrinsic and extrinsic pathways. However, as it has been found that the extrinsic pathway appears to be more critical for clotting (you don't bleed to death if you're missing Factor XII in the intrinsic pathway), we will focus on the extrinsic pathway.

The extrinsic pathway cascade kicks off when tissue factor (in the blood vessel wall) binds to Factor VIIa to form a FVIIa-TF complex. (Note: "a" just means that it's the activated form of Factor VII.) FVIIa-TF then goes on to activate other clotting proteins, ultimately leading to the production of thrombin (a.k.a. Factor IIa). Thrombin has many purposes. Firstly, it can convert fibrinogen to fibrin, which then goes on to stabilise the platelet plug. Secondly, it can catalyse the activation of Factor XIII, which is needed to cross-link fibrin and make it stronger. Finally, thrombin can catalyse the activation of clotting factors earlier in the pathway, including clotting factors in the intrinsic pathway (which may explain why you won't bleed to death if you're missing Factor XII, which is the first clotting factor in the intrinsic pathway). Thus, thrombin helps to propagate the cycle of clotting.

Fibrinolysis: Breakdown of the fibrin clot

Obviously, you need to be able to stop the clotting process somehow, or our vascular system would just become one giant clot every time we got a papercut. The process of breaking down a clot when it is no longer needed (or preventing new clots from taking hold) is called fibrinolysis. In fibrinolysis, tissue plasminogen activator (tPA) converts plasminogen to plasmin. Plasmin can attack fibrin, breaking it down to fibrin degradation products (FDPs), which can be cleared by macrophages.

Tests of Haemostasis

Bleeding History

Taking a good history is very important, as the main tests that we use (PT and APTT, which I'll explain in a bit) don't test everything. For instance, they don't test for Factor XIII (the clotting factor that cross-links fibrin). Therefore, if the tests are normal, but the patient reports a history of bleeding profusely after getting their wisdom teeth out, you should still be very careful!

Platelet Count

A platelet count is just that: a count of how many platelets you have. We normally have around 150-400 * 10^9 platelets per litre of blood. We have a massive amount of redundancy here, so most people don't show symptoms of thrombocytopaenia (low platelets) until their platelets get down to around 10 * 10^9/L or so. Low platelets may result in a rash that looks like small red dots: these dots are called petechiae (if they're small) or purpura (if they're a little larger) (at least that's what this website says).

PT/APTT

PT (Prothrombin Time) and APTT (Activated Partial Thromboplastin Time) measure the extrinsic and intrinsic pathways, respectively. (Remember, you need to "insert" an extra T to get the test that measures the "intrinsic" pathway.) Some factors are common to both pathways (II, V, and X) and thus affect the results of both tests. For both tests, an activator (tissue factor for measuring PT, some kind of "contact factor" such as Kaolin for measuring APTT) is added to centrifuged blood, along with a platelet substitute and calcium, and the amount of time that it takes to form a clot is measured. A long length of time to clot suggests that there is a problem in the pathway that is being measured.

Since the tissue factor used in PT might not be exactly the same in each lab, a standardised measure called the INR (International Normalised Ratio) was developed. The INR is basically a ratio of the PT of the patient sample compared to the PT of a normal sample. INR is good for monitoring patients over time, though there are some areas where PT might do a better job.

Fibrinogen

Fibrinogen can be measured using immunological or functional methods. Immunological methods measure the concentration of fibrinogen (presumably by using anti-fibrinogen antibodies or something similar), but not the functional activity. To measure the functional activity, you need to use a functional method, such as the Clauss assay, in which diluted plasma is mixed with a high concentration of thrombin. The plasma is kept dilute so as to remove the effects of inhibitory substances that might be in the plasma (e.g. heparin), and the thrombin concentration is kept high so that it is clear that any deviation in clotting time is not due to a lack of thrombin.