Biochemistry, Clotting Factors

Fundamentals

Hemostasis

Clotting factors are arguably the crux and most essential components of hemostasis. Hemostasis is the body’s physiologic response to vascular endothelial injury, which results in a series of processes that attempt to retain blood within the vascular system through the formation of a clot. Hemostasis can be further divided into primary and secondary hemostasis. Primary hemostasis, which results in the formation of a soft platelet plug, involves vasoconstriction, platelet adhesion, platelet activation, and platelet aggregation. Secondary hemostasis is primarily defined as the formation of fibrinogen into fibrin, which ultimately evolves the soft platelet plug into a hard, insoluble fibrin clot. Within primary and secondary hemostasis, 3 coagulation pathways exist: intrinsic, extrinsic, and common.[1][2][3][4]

Pathways

The intrinsic pathway responds to spontaneous, internal damage of the vascular endothelium, whereas the extrinsic pathway becomes activated secondary to external trauma. Both intrinsic and extrinsic pathways meet at a shared point to continue coagulation, the common pathway. Clotting factors involved in the intrinsic pathway include factors XII, XI, IX, and VIII. Clotting factors involved in the extrinsic pathway include factors VII and III. The common pathway includes clotting factors X, V, II, I, and XIII. Clotting factors can also be referred to outside of their Roman numeral designations. In the intrinsic pathway, factors XII, XI, IX, and VIII are also known as Hageman factor, plasma thromboplastin antecedent, Christmas factor, and antihemophilic factor A, respectively. In the extrinsic pathway, factors VII and III are also known as stabilizing factor and tissue factor, respectively. The common pathway factors X, V, II, I, and XIII are also known as Stuart-Prower factor, proaccelerin, prothrombin, fibrinogen, and fibrin-stabilizing factor, respectively. Clotting factor IV is a calcium ion that plays an important role in all 3 pathways. Some of the clotting factors function as serine proteases, specifically factors II, VI, IX, and X.

Cellular Level

The overwhelming majority of clotting factors are manufactured principally in hepatocytes. Hepatocytes are responsible for providing the body with clotting factors XIII, XII, XI, X, IX, VII, V, II, and I. Clotting factors VIII (antihemophilic factor A) and III (tissue factor) originate from endothelial cells, whereas clotting factor IV (calcium ion) is freely available in plasma. Megakaryocytes produce the body’s platelets and also contribute to the production of factor V.[5][6]

Mechanism

Vascular injury results in the exposure of subendothelial collagen and von Willebrand factor (vWF). vWF is a glycoprotein that serves as the initial stationary foundation on which a clot forms. Subendothelial vWF, which is also present in the vasculature and acts to increase the half-life of VII, binds to glycoprotein Ib (GpIb) on platelets. This causes a conformational change on the platelet surface that results in the exposure of glycoprotein IIb/IIIa (GpIIb/IIIa). Due to the conformational change, circulating fibrinogen attaches to GpIIb/IIIa. At this point in hemostasis, a soft platelet plug has formed, and the importance of biochemical interactions of clotting factors arises.

Membrane Binding

In addition to the exposure of GpIIb/IIIa as a result of conformational change occurring on the platelet, phosphatidylserine also emerges on the platelet surface. Phosphatidylserine is a membrane phospholipid whose polar end has a negative charge and, as a result, provides an excellent surface for a calcium ion to bind. The interaction between negatively charged phosphatidylserine and calcium does not completely negate calcium’s positive charge. This allows for serine proteases to bind to the surface of the platelet membrane. This binding is possible due to the carboxylation of clotting factors II, VII, IX, and X. These clotting factors have a region called gamma-carboxyglutamic acid that undergoes vitamin-K dependent carboxylation via gamma-glutamyl carboxylase. The enzyme adds a negatively charged carboxyl group to glutamic acid residues, which calcium easily binds to. As a result, the clotting factors can adhere to the platelet surface as serine proteases.

Intrinsic Pathway Proteases

Factor XII activation is the first step of the intrinsic pathway. Its activation is induced via contact with subendothelial collagen in the presence of high molecular weight kininogen. Graphically, zymogen to enzyme activation was denoted with the letter a, for example, XIIa. XIIa, in turn, activates XI into XIa, which leads to the activation of IXa. At this point, our previous discussion of gamma-carboxylation and platelet membrane interaction becomes important. Clotting factor IX plays its role as a serine protease within the intrinsic pathway. Although IXa is in its active form, IXa enzyme efficiency is abysmal without its essential cofactor, factor XIII. Once XIII and IXa are bound together (XIII-IXa) on the platelet membrane, proteolysis ensues. Specifically, the serine protease cleaves certain C-terminal arginine residues in the zymogen, which results in its subsequent activation. From here, we can understand how VIII-IXa activates factor X into Xa and leads to the common pathway.

Extrinsic Pathway Protease

Although the extrinsic pathway involves fewer steps than the common pathway, the role of serine proteases is just as important. When external insult occurs, clotting factor VII, along with its cofactor tissue thromboplastin, becomes an active protease and catalyzes X into Xa, which leads to the common pathway.

Testing

Prothrombin time (PT) measures coagulation throughout the extrinsic pathway and common pathway. A normal PT time is between 11 to 15 seconds; however, this time may vary slightly in the healthcare setting. The international normalized ratio (INR) is used to mitigate the slight discrepancies in PT and also is the test of choice when a patient is on warfarin therapy. A therapeutic INR is usually considered between 2 to 3 (for most clinical situations requiring anticoagulation with warfarin).[7][8][9]

Partial thromboplastin time (PTT) measures coagulation throughout the intrinsic pathway and common pathway. A normal PTT time is 25 to 40 seconds. PTT is the test of choice when monitoring a patient on unfractionated heparin. Of note, routine PTT surveillance is not necessary for patients on low-molecular-weight heparin.

Bleeding time (BT) is a measure of platelet function and how well platelets can form a clot. Normal bleeding time is 2 to 7 minutes. BT time is typically elevated in conditions of platelet dysfunction.

Pathophysiology

Here we will discuss commonly tested areas in regard to the pathophysiology of clotting factors.

Hemophilias

Hemophilia A is an X-linked recessive coagulopathy that results in dysfunctional VIII. From our earlier discussion, we can see how dysfunctional VIII will result in coagulopathy and prolonged PTT. Patients with this disorder will often present with easy bruising, bleeding after dental procedures or from operations in general, and hemarthrosis. Hemophilia A can be treated with desmopressin and recombinant factor VIII. Desmopressin causes endothelial cells to release vWF, which stabilizes VIII.

Hemophilia B, sometimes referred to as Christmas disease, is an X-linked recessive coagulopathy that results in dysfunction of IX. As with hemophilia A, hemophilia B will also cause a prolonged PTT. The difference is hemophilia A is a cofactor deficiency while hemophilia B is a protease deficiency; therefore, desmopressin will not be a good treatment option as these patients require recombinant factor IX. Hemophilia B will present with the same symptoms as hemophilia A. It is important to note that hemophilia A and B will have a normal PT/INR.

Von Willebrand Disease

vWF disease is the most commonly inherited coagulopathy. vWF can be differentiated from hemophilias in several ways. First, vWF mode of inheritance is autosomal dominant. Secondly, vWF disease is a disease of platelet dysfunction and will manifest primarily as mucosal membrane bleeding, such as epistaxis and prolonged menstrual cycles. Thirdly, bleeding time is normal in hemophilia, whereas it is prolonged in vWF disease. Since vWF increases the half-life of VII, you can expect to see a prolonged PTT in this disorder as well. Desmopressin can be used as a treatment option; however, certain subtypes of vWF disease do not warrant this treatment option. 

Vitamin K Deficiency

Previously, we discussed the importance of vitamin K and its clotting factors II, VII, IX, X, protein C, and S. The effects of vitamin K deficiency can be observed in both the extrinsic and intrinsic pathways and directly measured via PT and PTT, which will be prolonged. The etiology of vitamin K deficiency is extensive but commonly arises on test questions in regards to patients with poor diet, pancreatic insufficiency, liver disease, intestinal flora imbalances, neonates, or mimicked by patients on warfarin therapy.

Warfarin

Vitamin K assists in the carboxylation of clotting factors II, VII, IX, X, protein C and S. The enzyme responsible for gamma-carboxylation is vitamin K epoxide reductase, which is inhibited by warfarin. As mentioned previously, patients on warfarin will have the coagulation status measured via INR. In emergency clinical settings, warfarin’s therapeutic effects are negated by the administration of fresh frozen plasma. In a less urgent clinical setting, patients may be administered vitamin K. Rarely; patients may experience warfarin-induced skin necrosis within the first few days of beginning warfarin. This is due to protein C having the shortest half-life of vitamin K dependent clotting factors, and therefore a patient enters a prothrombotic state. However, this rare complication is more common in patients with protein C deficiency. To help eliminate this complication, patients are often co-administered heparin while beginning warfarin therapy as heparin’s onset is immediate while warfarin’s onset takes 2 to 3 days.

Clinical Significance

By understanding the biochemistry of clotting factors, healthcare professionals can quickly identify probable causes of a patient's coagulopathy by examining a patient's coagulation studies. For elevations in PT/INR, we can focus in on conditions such as liver disease, warfarin use, vitamin-K deficiency, and deficiencies in the extrinsic or common pathway. For elevations in PTT, we narrow our focus on more common causes such as hemophilias, unfractionated heparin use, vitamin-K deficiency, and vWF, with careful attention to BT. It is important to remember that prolongations in PT and PTT could also be due to deficiencies in the common pathway, but the previous conditions and examples yield a higher probability of identifying the root of the coagulopathy.[1][10]