Hemostasis

Hemostasis refers to the innate, stepwise body processes that occur following vessel injury, resulting in clot formation and cessation of bleeding. Hemostasis occurs in 2 phases, namely, primary and secondary. Primary hemostasis involves platelet adhesion, activation, and aggregation to the damaged vascular endothelium, forming a plug that stops the bleeding temporarily. Secondary hemostasis involves the activation of the coagulation cascade resulting in the formation of a more stable plug. Finally, as the vasculature is repaired, the clot is broken down in the fibrinolytic phase.

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Definition and Phases

Definition

Hemostasis refers to the innate, stepwise body processes that occur following vessel injury, resulting in clot formation.

Phases of the hemostatic process

  1. Constriction of the blood vessel: to limit blood flow to the area
  2. Formation of the platelet plug: the initial, temporary plug
  3. Activation of the coagulation cascade: to form a more stable fibrin clot
  4. Fibrinolytic phase: to break down the clot once it is no longer necessary

Vasoconstriction and Formation of the Platelet Plug

Injured vessels vasoconstrict after endothelial injury. Additionally, exposure of blood to the subendothelial components triggers formation of the platelet plug.

Vasoconstriction

Endothelial injury results in a transient vasoconstriction via:

  • Neural stimulation reflex: innate contraction of the vascular smooth muscles upon injury
  • Endothelin: a vasoconstrictor secreted from the damaged endothelial cells
  • Thromboxane: a vasoconstrictor released from platelets

Steps in formation of the platelet plug

Following an endothelial cell injury, the following processes occur with the platelets to form a temporary platelet plug (also known as primary hemostasis):

  • Adhesion
  • Activation
  • Aggregation
  • Secretion

Formation of the temporary hemostatic plug:
The disrupted endothelial surface exposes von Willebrand factor (vWF) to the passing blood. Platelets bind to vWF via their GpIb receptors and are activated. Platelet activation triggers the secretion of ADP, which stimulates the expression of the GpIIb/IIIa receptors on the platelets. The GpIIb/IIIa receptors bind to fibrinogen and a platelet on each end, causing platelets to aggregate. As more platelets bind to each other, a platelet plug is formed. As the coagulation cascade is activated, thrombin converts the weaker fibrinogen into the stronger fibrin, creating a much more stable clot.

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Platelet adhesion

Exposure of the blood to subendothelial components at the site of injury causes platelets to adhere to the injury site.

  • GpIb receptors on the platelets bind to the exposed von Willebrand factor (vWF) within the subendothelial matrix. This bond is strong enough to withstand the shearing force of the flowing blood.
  • Other adhesion interactions occur:
    • Involve collagen, other glycoprotein receptors, and tyrosine kinase receptors
    • Contribute to both adhesion and activation of platelets
  • Adherent platelets are activated.

Platelet activation

Activated platelets enhance further platelet adhesion and aggregation, and stimulate secretion. 

  • Platelet activators:
    • Potent platelet activators:
      • Thrombin: produced in the coagulation cascade
      • Collagen: interacts with platelets at the site of injury
    • Weaker platelet activators:
      • ADP: acts in an autocrine fashion → released by platelets to help activate other platelets
      • Epinephrine
  • Activated platelets:
    • Undergo shape change to become an elongated pseudopod → new shape is extremely adherent
    • Activate their GpIIb/IIIa receptor so that they are capable of binding to fibrinogen
    • Release their granules (see “Platelet secretion” below) → assists in activation of the coagulation cascade

Platelet aggregation

  • GpIIb/IIIa receptors present on the activated platelets begin binding to fibrinogen.
  • Fibrinogen is a symmetrical molecule that can bind 2 platelets simultaneously (1 on each end of the fibrinogen).
  • Forms fibrinogen bridges between platelets
  • Results in platelet aggregation and formation of a primary hemostatic plug

Platelet secretion

Platelets contain 2 types of granules. These granules release various substances when platelets are activated. 

  • Functions of secreted substances:
    • Recruit and activate additional platelets
    • Stimulate expression of GpIIb/IIIa on platelets → enhanced aggregation
    • Promote vasoconstriction
    • Stimulate the process of vascular repair via fibroblast/smooth muscle cell recruitment
    • Contribute to initiation of the coagulation cascade
  • Alpha granules contain:
    • Fibrinogen
    • vWF
    • Factor V (part of the common pathway of the coagulation cascade)
    • Platelet-derived growth factor (PDGF)
    • Platelet factor-4 
    • Fibronectin
    • Thrombospondin
  • Dense granules contain:
    • ADP 
    • Serotonin
    • Histamine
    • Calcium 

Coagulation Cascade

Overview

The coagulation cascade is a series of reactions that ultimately generates a strong, cross-linked fibrin clot. This process is also known as secondary hemostasis.

  • A number of coagulation factors undergo sequential activation by 1 of the 2 pathways:
    • Extrinsic pathway: primarily responsible for initiation of the cascade
    • Intrinsic pathway: primarily involved in amplification of the cascade
  • Common pathway:
    • The extrinsic and intrinsic pathways join together to form the final common pathway when factor X is activated.
    • Formation of the fibrin clot occurs at the end of the common pathway.
  • Initiation:
    • The extrinsic pathway is activated with endothelial injury and ultimately produces activated factor X (Xa).
    • Factor Xa then moves through the common pathway.
    • Thrombin is produced in the common pathway.
  • Amplification:
    • The initial production of thrombin activates multiple factors in the intrinsic and common pathways.
    • As the intrinsic pathway is activated, an increased amount of factor Xa is produced.
    • Factor Xa allows for increased activation of the common pathway:
      • More fibrin is produced → propagates the clot
      • More thrombin is produced → positive feedback loops

Overview of the coagulation cascade
a: activated form
PF3: platelet factor 3 (phospholipids)

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Coagulation factors

Coagulation factors are trypsin-like serine proteases and are denoted with roman numerals.

  • All procoagulant factors are synthesized in the liver except:
    • Factor VIII: produced in endothelial cells 
    • vWF: produced in the megakaryocytes and endothelial cells
  • Vitamin K-dependent factors: 
    • Undergo carboxylation to become functional, require vitamin K
    • Procoagulants:
      • Factor II 
      • Factor VII
      • Factor IX
      • Factor X
    • Anticoagulants:
      • Protein C
      • Protein S
    • Vitamin K:
      • Primarily synthesized in the colon
      • Activated by epoxide reductase in the liver
      • Works as a cofactor for gamma-glutamyl carboxylase to carboxylate the Vitamin-K dependent factors
      • These carboxylated factors gain affinity for the negatively charged phospholipids on platelets → promote coagulation
  • Form multicomponent enzyme complexes which:
    • Perform critical steps in the coagulation cascade
    • Each contain a protease, a cofactor, and a substrate
    • Bound to anionic phospholipid membrane surfaces
      • Restrict a majority of thrombin generation to the sites of vascular injury
  • 3 primary procoagulant multicomponent enzyme complexes:
    • Extrinsic X-ase (pronounced tenase): 
      • Factor VIIa (protease) + tissue factor (cofactor) + factor X (substrate)
      • Activates factor X → factor Xa
    • Intrinsic X-ase:
      • Factor IXa (protease) + factor VIIIa (cofactor) + factor X (substrate)
      • Activates factor X → factor Xa
    • Prothrombinase:
      • Factor Xa (protease) + factor Va (cofactor) + prothrombin (substrate)
      • Activates prothrombin → thrombin

Extrinsic pathway: The tissue factor pathway

The extrinsic pathway is the primary physiological mechanism by which clotting is initiated.

  • Involves extrinsic X-ase
  • Begins with the tissue factor in the exposed subendothelial matrix:
    • A membrane glycoprotein
    • Expressed only after endothelial injury
  • Tissue factor activates factor VII → VIIa
  • Factor VIIa activates factor X → Xa. Factor Xa is the 1st step in the common pathway.
  • To summarize, tissue factor activates VII → VIIa, which activates X → Xa → common pathway

Intrinsic pathway: The contact pathway

The intrinsic pathway is mainly responsible for the amplification of factor X activation. Factor X is activated by the initial thrombin generated by the extrinsic/common pathway, but also can be activated directly by endothelial injury.

  • Exposure to negatively charged collagen in the subendothelial matrix activates high-molecular-weight kininogen (HMWK) and prekallikrein (PK).
  • HMWK + PK activate factor XII → XIIa
  • Factor XIIa activates:
    • Factor XI → XIa
      • Thrombin (from the common pathway) also activates factor XI.
    • Prekallikrein → kallikrein
      • Kallikrein augments further activation of XII → XIIa
  • Factor XIa activates factor IX → IXa
  • Intrinsic X-ase: factor IXa (protease) combines with factor VIIIa (cofactor) to activate factor X (substrate) → Xa
    • Factor VIII:
      • Activated by factor Xa and thrombin (both initially generated by the extrinsic and common pathways)
      • Stabilized by vWF
    • Factor Xa is the 1st step in the common pathway.
  • To summarize, HMWK + PK activate → 12, which activates → 11, which activates → 9, which combines with 8 to activate → 10

The extrinsic and intrinsic coagulation systems

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Common pathway

  • Begins with prothrombinase: factor Xa combines with factor Va and calcium to activate prothrombin (factor II) → thrombin (factor IIa)
  • Thrombin (factor IIa) activates the following:
    • Fibrinogen (factor I) → fibrin (factor Ia) → clot propagation
    • Factor XIII → XIIIa → cross-linking of fibrin polymers to stabilize the clot
    • Factor XI → XIa in the intrinsic pathway
    • Factor VIII → VIIIa in the intrinsic pathway
    • Factor V → Va in the common pathway
    • Platelets → activated platelets → aggregation and secretion
  • Generation of thrombin leads to multiple positive feedback loops → ↑↑ production of thrombin

The final common pathway
a: activated form
PF3: platelet factor 3 (phospholipids)

Inhibition of Clotting and the Fibrinolytic Phase

Inhibition of clotting

The body produces several substances that inhibit platelet binding, aggregation, and secretion, as well as function as natural anticoagulants. These mechanisms limit clotting to specific focal sites and keep the blood fluid.

  • Tissue factor pathway inhibitor (TFPI):
    • Inhibits the activation of factor X
    • Located primarily on the surface of microvascular endothelial cells
  • Antithrombin:
    • Natural circulating anticoagulant produced by the liver
    • Inhibits activated forms of factors II, IX, and X
    • Rate of factor inactivation is augmented by heparin
  • Proteins C and S:
    • Vitamin K-dependent factors produced by the liver 
    • Protein C cleaves and inactivates factors V and VIII.
    • Protein S augments the activity of protein C.
  • Other anticoagulant substances produced by endothelial cells:
    • Prostacyclin: a vasodilator that blocks platelet aggregation
    • Nitric oxide: a vasodilator that blocks platelet adhesion and aggregation
    • Thrombomodulin: binds to thrombin and converts it into an anticoagulant that activates protein C

Fibrinolytic phase

The fibrinolytic system functions to remove the clot after the vasculature is repaired, and the process is accomplished primarily by plasmin.

  • Plasmin: cleaves fibrin polymers (fibrinolysis) 
  • Plasminogen is activated (converted to plasmin) by:
    • Tissue plasminogen activator (TPa)
    • Urine plasminogen activator (UPa) aka urokinase
    • Both are secreted by endothelial cells.
  • Fibrinolysis:
    • Forms fibrin-degradation products (e.g., D-dimer)
    • Generates new plasmin-binding sites on partially degraded fibrin

Laboratory Evaluation of Hemostasis

  • PT: 
    • Time taken for the plasma to clot when exposed to tissue factor
    • Measures function of the extrinsic and common pathways
    • Normal range: approximately 11–13 seconds
    • Elevated in:
      • Warfarin therapy
      • Vitamin K deficiency
      • Deficiency of factors II, V, VII, and X
      • Liver disease 
      • Disseminated intravascular coagulation (DIC)
  • INR:
    • A ratio comparing the patient’s PT to a reference PT
    • Measures function of the extrinsic and common pathways
    • Normal range: approximately 0.8–1.1
  • PTT: 
    • Time taken for the plasma to clot when exposed to a negatively charged substance (which activates the intrinsic pathway)
    • Measures function of both the intrinsic and common pathways
    • Normal range: 25–40 sec
    • Elevated in:
      • Heparin therapy 
      • Hemophilia (abnormal factor VIII or IX)
      • von Willebrand disease (vWD)
      • Liver disease
      • DIC
  • Bleeding time (BT):
    • Measures platelet function
    • Normal range: 2–7 minutes
    • Prolonged in:
      • Thrombocytopenia
      • DIC
      • vWD
      • Bernard-Soulier disease
      • Glanzmann’s thrombasthenia
      • Renal failure
      • NSAID and/or aspirin use
  • Fibrinogen:
    • Precursor to fibrin
    • Abnormally low levels can increase bleeding risk.
    • Normal range: 200–400 mg/dL
    • Abnormal bleeding tends to occur when levels < 100 mg/dL
  • D-dimer:
    • A primary fibrin-degradation product
    • Released upon cleavage of cross-linked fibrin by plasmin
    • Indicates recent or ongoing coagulation and fibrinolysis
    • Normal range: < 500 ng/mL

Normal hemostasis laboratory evaluation

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Clinical Relevance

Disorders of primary hemostasis (formation of the platelet plug)

  • Glanzmann thrombasthenia: an autosomal recessive bleeding syndrome characterized by a deficiency of the GpIIb/IIIa receptor, resulting in a lack of platelet aggregation
  • Bernard-Soulier syndrome: an autosomal recessive bleeding syndrome characterized by deficiency of the GpIb receptor, resulting in failure of platelet adhesion. Bernard-Soulier syndrome can be diagnosed using a ristocetin assay. Ristocetin activates vWF to allow binding to the platelet GpIb receptor; however, in Bernard-Soulier syndrome, the platelets will fail to adhere in the assay.
  • Immune thrombocytopenia: an autoimmune disorder characterized by anti-GpIIb/IIIa auto-antibodies, which lead to the destruction of platelets. Immune thrombocytopenia often occurs after GI or respiratory viral infections, although it can also be a drug-induced condition. Clinically, immune thrombocytopenia may present with prolonged bleeding, petechiae, easy bruising, and/or purpura. Treatment may include a platelet transfusion or splenectomy, or management with steroids and IV immunoglobulins.
  • Thrombotic thrombocytopenic purpura (TTP): a bleeding disorder marked by a pentad of fever, microangiopathic hemolytic anemia, thrombocytopenia, renal failure, and neurological symptoms. Thrombotic thrombocytopenic purpura occurs due to a congenital or acquired deficiency of ADAMTS-13, which is a metalloprotease that cleaves the vWF. A deficiency of ADAMTS-13 results in large vWF multimers that increase platelet aggregation, leading to microvascular thrombosis and a consumption of platelets.

Disorders of secondary hemostasis (the coagulation cascade)

Hemophilia: a rare blood-clotting disorder in which the body lacks blood-clotting factors (factor VIII in hemophilia A; factor IX in hemophilia B). Affected individuals present with abnormal bleeding that can occur spontaneously or after minor trauma. These individuals can bleed into joint spaces and develop life-threatening internal bleeding.

Mixed disorders affecting both platelets and coagulation factors

  • vWD: the most commonly inherited disorder of hemostasis caused by a qualitative or quantitative deficiency of von Willebrand factor. There are 3 primary types, which differ in severity, although all tend to present with bleeding abnormalities. Von Willebrand factor is required for both initial platelet adhesion and to help stabilize factor VIII in the intrinsic pathway. 
  • DIC: a serious medical condition in which the coagulation cascade is activated systemically, leading to multiple clots that can lead to permanent end-organ damage. During DIC, the coagulation factors are completely consumed. Disseminated intravascular coagulation always has a secondary cause. Infections, burns, and malignancies are among the most common causes. Disseminated intravascular coagulation can also occur during a severe postpartum hemorrhage. Laboratory findings include thrombocytopenia, prolongation of PT and PTT, and elevation of D-dimer levels.
  • Cirrhosis: The liver is the primary site of synthesis for a majority of the clotting factors. In addition to the impaired synthesis of clotting factors, cirrhosis may also independently result in thrombocytopenia due to splenic sequestration of platelets and decreased thrombopoietin production. Platelets themselves may also be dysfunctional.

Disorders of fibrinolysis

  • Factor V Leiden mutation: results in the production of mutant factor V, which is resistant to degradation by activated protein C, thereby leading to increased thrombin production and a procoagulant state in the blood. Complications include deep vein thrombosis, cerebral vein thrombosis, and pulmonary embolism.
  • Prothrombin gene mutation: the 2nd most common inherited thrombophilia after factor V Leiden. Point mutations in the prothrombin gene lead to increased levels of prothrombin, leading to a hypercoagulable state and increased risk of venous thromboembolism.
  • Protein C or S deficiency: results in the failure to inactivate factors Va and VIIIa. Like factor V Leiden, there is an increased risk for venous thromboembolism and warfarin-induced skin necrosis.
  • Antithrombin deficiency: an inherited or acquired disorder resulting in antithrombin activity that is < 80% of its normal activity. Antithrombin deficiency leads to a decreased inhibition of factors II, IX, and X, thus, creating a hypercoagulable state.

References

  1. Leung, L. (2019). Overview of hemostasis. In Tirnauer, J.S. (Ed.), UpToDate. Retrieved March 13, 2021, from https://www.uptodate.com/contents/overview-of-hemostasis
  2. Zehnder, J.L. (2020). Clinical use of coagulation tests. In Tirnauer, J.S. (Ed.), UpToDate. Retrieved March 27, 2021, from https://www.uptodate.com/contents/clinical-use-of-coagulation-tests 
  3. Longo, Dan; Fauci, Anthony; Kasper, Dennis; Hauser, Stephen; Jameson, J.; and Loscalzo, Joseph. Harrisons Manual of Medicine, 16th Edition. US: McGraw-Hill Professional, 2012. Pgs. 337–340.

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