The classical extrinsic/intrinsic model of blood coagulation is a bit medieval. It’s useful for understanding how coagulation factors might interact in simple terms, and helps us understand coagulation testing systems that are based on that way of thinking. However, it doesn’t accurately describe just how coagulation factors and platelets work together to form a clot. This process is often divided (in another man-made attempt at simplification) into primary and secondary haemostasis:

  • Primary haemostasis is platelet adhesion, activation and aggregation.
  • Secondary haemostasis is the formation of a fibrin clot around the platelets.

The endothelium usually inhibits coagulation

A healthy endothelium presents many challenges to blood clotting. Firstly, it releases two key molecules that inhibit coagulation: nitric oxide (NO) and prostacyclin (AKA prostaglandin I2). Both NO and prostacyclin inhibit platelet adhesion and activation, and are also vasodilators. The endothelium also expresses heparan, to which antithrombin binds, ready to inactivate many of the coagulation cascade factors. Finally, Tissue Factor Pathway Inhibitor (TFPI) binds to the endothelium, ready to inhibit blood coagulation from the very beginning. These inhibitory checks in the system prevent haemostasis from occurring inappropriately under normal conditions.

von Willebrand Factor and Tissue Factor

The endothelium is Janus-faced. One the one hand, it usually inhibits coagulation; on the other it can actively participate in the process when appropriately stimulated (e.g. by thrombin). Endothelial cells constitutively synthesise and release von Willebrand factor (vWF) into blood, which serves two functions (at least). Firstly, vWF stabilises coagulation factor VIII and protects it from proteolytic degradation in plasma. Secondly, vWF binds avidly to collagen and activates platelets when it does so. Now, blood and collagen don’t usually come into contact with each other unless the endothelium is damaged. When the endothelium is disrupted – say by a cut through a vessel – vWF immediately binds to collagen ready to activate the first platelet that comes along. People with von Willebrand disease have mutations to vWF which affect its function leading to bleeding disorders. The endothelium not only idly secretes a little vWF, it stores some away in Weibel-Palade bodies ready to be explosively released when required (Figure 1).

Tissue Factor (TF) is expressed by most subendothelial cells and activates the extrinsic pathway for blood coagulation when it becomes exposed by damage to endothelial cells (Figure 2). When robustly activated - as in inflammatory conditions - the endothelium can also become stimulated to express TF. The changing face of the endothelium from anti-coagulant to pro-coagulant can lead to disseminated intravascular coagulation in diseases where inflammation is widespread, such as in sepsis.

Platelet adherence, activation and aggregation

After damage to the endothelium, vWF binds to collagen and to vWF receptors on platelets, causing them to adhere to the site of damage and beginning the process of platelet activation and aggregation (Figure 1). During the activation process, platelets release other mediators, such as thromboxane A2 (TXA2) and adenosine diphosphate (ADP) which activate other nearby platelets (Figure 2). Thrombin can also activate platelets, as can collagen itself (although this seems to be less important than vWF). Once activated, platelets express fibrinogen receptors on the cell surface. Fibrinogen acts as a linker between platelets, causing them to aggregate.

Figure 1: ❶The endothelium continuously releases small amounts of von Willebrand Factor, which circulates in the blood. Endothelial cells also store von Willebrand Factor in Weibel-Palade bodies for release when appropriately stimulated. ❷If collagen becomes exposed to blood (because the endothelium is damaged), von Willebrand Factor binds to it. ❸Platelets express receptors for both collagen and von Willebrand Factor (although von Willebrand Factor seems to be more important) and become activated when these proteins bind to them. Activated platelets change shape and express functional fibrinogen receptors, which are required for aggregation.

”It seems like a lot of things activate platelets!”

Yes, you don’t want platelet activation and clotting to fail when it is required, so multiple trigger mechanisms have evolved. Scientists who study platelet aggregation often use a simple in vitro model to examine the effects of platelet-aggregating substances on isolated platelets. This requires the preparation of platelet-rich plasma (PRP) from whole blood using several steps of centrifugation to remove other blood cells.

To study platelet aggregation, a glass tube of PRP is placed in a platelet aggregometer (yes, really), which shines a fixed beam of light through the sample. If platelets aggregate, the solution becomes less cloudy (because the platelets clump together) and the transmittance of light increases. Using this simple system it is easy to demonstrate that – on their own – thrombin, collagen, TXA2 or ADP (amongst others) can cause platelet aggregation. What happens in the body is probably a bit of everything.

Figure 2: ❶ Once activated, platelets begin to aggregate by binding to fibrinogen, which links them together. At the same time, platelets release multiple pro-activation/aggregation signalling molecules such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2). The activation and aggregation of platelets is often termed primary haemostasis. ❷ Tissue factor (TF), expressed by nearly all sub-endothelial cells activates the coagulation cascade to initiate a minor burst of haemostasis. Factor FVIIa binds to Tissue Factor, and goes on to activate Factor IX, which activates thrombin from prothrombin ❸. Thrombin activates receptors on platelets as well as the endothelium, amplifying platelet aggregation and initiating release of stored von Willebrand Factor from endothelial cells.

Secondary haemostasis: fibrin formation

Platelet activation and aggregation result from activation of membrane receptors and occur in a matter of seconds, whereas blood clotting requires activation of multiple proteases over a longer timeframe. Once the damage to the endothelium occurs, subendothelial TF is exposed to the blood and the extrinsic coagulation pathway is activated. Tissue Factor acts as a receptor for FVII, which can be subsequently activated by many of the coagulation cascade enzymes, including itself. Once activated, FVIIa activates FXa, which subsequently cleaves thrombin from prothrombin (Figure 2). Initially, these reactions proceed at a fairly slow rate, but subsequently thrombin production is greatly amplified by the production of important cofactors (Figure 3). Thrombin is acts to activate platelets and endothelial cells, in addition to its role as the key protease in the coagulation cascade.

Figure 3: Thrombin activates two cofactors, Factor VIIIa ❶ and Factor Va ❷ which subsequently form calcium ion-dependent complexes on the surface of platelets with Factor Xa (AKA the prothrombinase complex) and Factor IXa (AKA the tenase complex). These complexes greatly accelerate production of Factor Xa and thrombin, respectively. This is the amplification stage of the coagulation cascade. Although there is probably some fibrin formation during the initial activation of thrombin via the TF pathway, the greatly increased production of thrombin via tenase and prothrombinase contributes considerably more to the process. Fibrin formation is often termed secondary homeostasis.

Amplification of thrombin production

Thrombin activates two important cofactors, FV and FVIII. Neither is a protease, but both require cleavage by thrombin to become activated. Using Ca2+ ions in plasma, these cofactors help bring together two complexes on the charges surfaces of activated platelets:

  • The tenase complex: FVIIIa+FIXa to rapidly generate FXa
  • The prothrombinase complex: FVa+FXa to rapidly generate active thrombin
The subsequent explosive production of thrombin is responsible for the formation of the insoluble fibrin mesh that holds a blood clot together.

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