Secondary Hemostasis

Note: Please refer to the diagram, Interactive Coagulation Cascade.

Secondary hemostasis refers to the cascade of enzymatic reactions that ultimately results in the conversion of fibrinogen to fibrin monomers. Fibrin monomers are then cross-linked into insoluble strands that serve to stabilize the loose platelet clot formed in primary hemostasis. Secondary hemostasis is triggered by the release of tissue factor from epithelial cells that are exposed to the circulation at the site of vascular injury. Defects in secondary hemostasis decrease fibrin production and reduce the stability of the formed clot.1-4 In these conditions, bleeding is generally delayed compared to that observed in defective primary hemostasis. The loose platelet plug is not stabilized by fibrin strands and starts to leak. Typical symptoms of patients with defective secondary hemostasis include soft-tissue bleeding, hematomas, retroperitoneal bleeding, or hemarthrosis. The hemophilias are examples of defects in secondary hemostasis.3

Two simple screening tests are most often used in the initial assessment of secondary hemostasis. Both the prothrombin time (PT) and partial thromboplastin time (PTT) are performed on plasma separated from whole blood collected with sodium citrate anticoagulant. Citrate binds calcium in the blood and inhibits clot formation. Like all other clot-based coagulation tests, both the PT and PTT assays are initiated by adding excess calcium to the testing tube to overcome the effect of the citrate. The testing sequence for PT and PTT are explained below.

In normal hemostasis, tissue factor that is released into the circulation as the result of vascular damage unleashes a coagulation cascade that ultimately results in the production of fibrin.1,3,5Phospholipid membranes play an integral role in providing a surface for catalytic enzyme complex formation for these reactions. Platelets that form the primary hemostatic plug provide the phospholipid required to promote fibrin generation.2 The PT test brings these components (tissue factor and phospholipid) together in the test tube. Since tissue factor is essentially absent in the normal plasma and must be supplied from an external source, the cascade of enzymatic reactions triggered in the PT test is referred to as the “extrinsic pathway.”

The term “partial thromboplastin” is derived from the fact that this assay differs from the PT (which uses “complete thromboplastin”) in that no tissue factor is used to initiate clotting.1,3,4 Instead, the reagent for the PTT simply includes a source of phospholipid. The clinical chemists who developed the PTT gave the name “intrinsic pathway” to the cascade of reactions triggered in the PTT test based on the mistaken perception that coagulation that occurs in the PTT test is initiated without the addition of any external factors. It is now understood that an external factor is, in fact, involved in the initiation of the PTT clotting cascade.6 The intrinsic pathway is actually activated by plasma contact with the negatively charged glass surface of the test tube. This “contact activation” can be enhanced by adding particulate matter like silica, kaolin, or ellagic acid into the test mixture in what we now refer to as an “activated” partial thromboplastin time, or aPTT.

The enzymatic reactions of the extrinsic and intrinsic pathways of coagulation are depicted in Figure 1. For many years clinical scientists felt that these two pathways, corresponding to the PT and aPTT laboratory tests respectively, represented unique physiologic mechanisms for the initiation of normal coagulation.1,3,7 It turns out, however, that while several steps of the intrinsic pathway are critical to normal hemostasis, contact activation is not a physiologically relevant mechanism for the initiation of normal coagulation. This became evident when it was discovered that even severe deficiencies of the intrinsic pathway factors (HMWK, prekallikrein, or factor XII) that will produce a markedly extended aPTT in vitro will not cause bleeding in vivo. It is now clear that extrinsic activation by tissue factor is the primary initiator of normal coagulation. Tissue factor forms a complex with activated VIIa that initiates the extrinsic pathway. This leads to the formation of thrombin, which in turn activates factor VIII. The tissue factor/factor VIIa complex also directly activates factor IX to IXa. Together, activated factors VIIIa and IXa form the potent “tenase” complex, effectively bypassing the contact activation pathway. Contact activation is just a handy trick that allows the clinical chemist to assess the functionality of several important intrinsic pathway factors in a test tube. Contact activation can occur in vivo when negatively charged surfaces, such as heart valve prostheses or vascular stents, are installed in the blood stream.5

Gamma-carboxyl glutamate (gla) residues on factors II, VII, IX, and X play a critical role in effective secondary hemostasis.1,7 These gla residues are formed by vitamin K-dependent enzymes and facilitate calcium-dependent complex-binding to phospholipid membranes. This serves to concentrate the catalytic activity at the point of injury and to prevent thrombosis from spreading throughout the vasculature. These complexes consist of a proteolytic enzyme associated with a catalytic cofactor that is anchored to a negatively charged phospholipid surface by gla residues. Vitamin K deficiency or inhibition of vitamin K-dependent enzymes by oral anticoagulants serves to block the formation of gla residues and disrupt hemostasis. Since factor VII, an extrinsic pathway factor, has the shortest half-life of the vitamin K-dependent cofactors, the PT test is more sensitive to oral anticoagulant therapy or vitamin K deficiency than the aPTT.4


1. Brandt JT. Overview of hemostasis. In McClatchey KD, ed. Clinical Laboratory Medicine. 2nd ed. Baltimore, Md: Lippincott Williams and Wilkins; 2002:987-1009.

2. Kottke-Marchant K, Corcoran G. The laboratory diagnosis of platelet disorders. Arch Pathol Lab Med.2002; 126(2):133-146. PubMed 11825107

3. Liu MC, Kessler CM. A systemic approach to the bleeding patient. In Kitchens CS, Alving BM, Kessler CM, eds. Consultative Hemostasis and Thrombosis. Philadelphia, Pa: WB Saunders; 2002:181-196.

4. Van Cott EM, Laposata M. Coagulation. In Jacobs DS, Oxley DK, DeMott WR, eds. Laboratory Test Handbook. Hudson, Ohio: Lexicomp; 2001:327-358.

5. Mann KG, Brummel K, Butenas S. What is all that thrombin for?” J Thromb Haemost. 2003; 1(7):1504-1514. PubMed 12871286

6. Adcock DM, Jensen R, Johns CS, et al. Coagulation Handbook. Austin, Texas: Esoterix Coagulation; 2002.

7. Triplett DA. Coagulation abnormalities. In McClatchey KD, ed. Clinical Laboratory Medicine. 2nd ed. Baltimore, Md: Lippincott Williams and Wilkins; 2002:1033-1049.


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