The Hemostatic Mechanism

Normal hemostasis involves a series of physiologic checks and balances that assure that blood remains in an invariably liquid state as it circulates throughout the body but, once the vascular network is violated, transforms rapidly into a solid state. The transformation into a solid state must then be complemented by a process to eliminate clot no longer needed for hemostasis. That process is fibrinolysis.

The understanding of the coagulation process has evolved over the past decade from one in which platelets and the triggering of two very separate protein cascade systems – the intrinsic and extrinsic pathways – would ultimately produce clot formation, to the present understanding that places more emphasis upon the final common pathway and the proteolytic systems that result in the degradation of formed clots and the prevention of unwanted clot formation.

Background

The classical dual-cascade model of coagulation proved to be inadequate because it failed to explain several clinical phenomena:

  1. Persons lacking factor XII, prekallikrein, or high-molecular-weight kininogen do not bleed abnormally, suggesting that contact activation is not critical for normal hemostasis.
  1. Patients with only trace quantities of factor XI withstand major trauma without unusual bleeding, and those completely lacking factor XI have only a mild hemorrhagic disorder. Factor XI therefore appears to have a more minor role in coagulation than ascribed to it by classical theory.
  1. Deficiencies of factor VIII (an extrinsic pathway factor) and factor IX (an intrinsic pathway factor) lead to Hemophilia A and B, respectively. The classical description of two pathways of coagulation leaves it unclear why either type of hemophiliac could not simply clot via the unaffected pathway.
  1. Most importantly, while the classical theories provide a reasonable model of in vitro coagulation tests, they fail to incorporate the central role of cell-based phospholipid surfaces in the in vivo coagulation process.

Coagulation – Current Concepts

  • Coagulation begins when vascular injury exposes blood to the subendothelial membrane-bound protein, tissue factor (TF), and to subendothelial collagen.
  • Activation phase: Exposed TF activates a series of factors, culminating in the formation of a “prothrombinase complex” on the phospholipid surface of the TF. The prothrombinase complex catalyzes the formation of a small amount of thrombin, which then serves as a priming mechanism for subsequent hemostatic events.
  • Exposed collagen in the injured vascular wall stimulates platelets to adhere to each other at the site of injury. In addition glycoprotein ligands on the platelet surface, which are not expressed at rest, are exposed as the platelets are activated.
  • GPIb receptors bind vonWillebrand factor (vWF), which attaches platelets to the subendothelial surface of the injured blood vessel.
  • GPIIb/IIIa receptors bind platelets together (aggregation), and also bind fibrin, which helps stabilize the platelet plug.
  • Acceleration phase: The thrombin generated by the TF-bound prothrombinase complex amplifies the coagulation process:
  • Further activation of platelets, resulting in:
  • The exposure of more glycoprotein receptors on the platelet surface, thus further platelet aggregation
  • Platelet degranulation, releasing a host of factors involved in the aggregation process and coagulation cascade, among which is ADP, a powerful platelet activator proaggregant that rapidly recruits additional platelets to the growing platelet mass.
  • Activation of Factor V, which catalyzes the conversion of prothrombin to thrombin
  • Release of factor VIII from its vWF carrier molecule and activation of that factor VIII
  • Activation of factor XI, which in turn activates factor IX, adding to the pool of activated factor IX first formed during the activation phase, and accelerating the formation of thrombin.
  • The net result of the acceleration phase is the availability of activated platelets and activated factors V, VIII, and IX.
  • Propagation phase: The platelets in the platelet plug then provide the phospholipid surface on which factor complexes form and produce an explosive generation of thrombin.
  • Thrombin thus generated catalyzes the formation of fibrin from fibrinogen. Fibrin acts to crosslink the platelets to reinforce the friable platelet plug.
  • Thrombin also activates thrombin activatable fibrinolysis inhibitor (TAFI) and factor XIII, both of which serve to stabilize the fibrin clot.
  • The cross-linked fibrin meshwork shrinks and traps activated platelets and red blood cells to form a strong clot.
  • Fibrinolysis: primarily involves the production of plasmin, which serves to remodel fibrin clots and recanalize thrombosed blood vessels.
  • Plasmin is formed by the conversion of plasminogen to plasmin.
  • When circulating plasminogen comes into contact with fibrin, it binds to that fibrin and becomes incorporated into the growing fibrin clot, along with tissue plasminogen activator (t-PA).
  • t-PA only activates plasminogen that is bound to fibrin.
  • Plasmin degrades fibrin to D-dimers and fibrin degradation products, which are normally cleared by the liver, kidney, and reticuloendothelial system. When produced in high concentrations, however, FDPs impair platelet function, inhibit thrombin, and prevent crosslinking of fibrin strands, resulting in a clot that is more readily degraded by plasmin.
  • The drugs -aminocaproic acid and tranexamic acid enhance clotting by inhibiting the conversion of plasminogen to plasmin.

Control of Coagulation – Protection From Spontaneous Coagulation Reactions

Coagulation must be precisely regulated to prevent uncontrolled clotting such as that which occurs with DIC. Several mechanisms are involved in the regulation and control of coagulation.

  • Endothelial inhibition – Intact vascular endothelium has properties that serve to limit both platelet aggregation and coagulation, and to induce fibrinolysis should a clot begin to form on normal endothelium.
  • Primary hemostasis is, in part, controlled by the balance between the actions of two prostaglandins, thromboxane A2 and prostacyclin.
  • Thromboxane A2 is synthesized at the site of vascular damage by activated platelets. TxA2 has two hemostatic effects:
  • It is a potent vasoconstrictor that causes blood vessels to constrict locally and shunt blood flow away from the site of injury.
  • It stimulates additional ADP release from platelets and thereby causes recruitment of additional platelets.
  • Prostacyclin (PGI2)is synthesized by normal vascular endothelium remote from the site of vascular injury and has actions opposite those of TxA2.
  • It is a potent vasodilator and helps to clear any activated clotting factors.
  • It inhibits platelet activation, secretion and aggregation.
  • It therefore serves to prevent platelet aggregation and clot formation on the endothelial surface beyond the site of injury.
  • Nitric oxide, synthesized by normal endothelium, is another vasodilator and inhibitor of platelet aggregation that potentiates the effect of prostacyclin.
  • ADP-ases, expressed on the surface of endothelial cells, degrade any excess ADP that might otherwise initiate platelet aggregation on normal endothelial surfaces.
  • Inhibition of Coagulation – Many factors serve to limit and localize clot formation.
  • Clotting factors circulate in an inactive form.
  • Once they do become activated, normal blood flow dilutes their concentration and washes them away from sites of injury, thus limiting clot formation.
  • Activated clotting factors are preferentially removed from the circulation by the liver and reticuloendothelial system.
  • Because some interactions of the coagulation pathway require the presence of a phospholipid surface, clot formation is localized to these phospholipid surfaces.
  • Specific Coagulation Inhibition Systems – Native Anticoagulants
  • Thrombin, thrombomodulin, protein C and protein S
  • Thrombin, by interacting with Protein C, a vitamin K dependent anti-coagulant protein, can, in an example of negative feedback, decrease its own synthesis by inhibiting factors V and VIII.
  • Thrombomodulin, a protein located on the vascular endothelial cell surface, binds thrombin and alters its molecular structure such that it can no longer activate factors V and VIII or catalyze the conversion of fibrinogen to fibrin.
  • The thrombin-thrombomodulin complex activates protein C, which in turn, along with protein S as a cofactor, cleaves activated factors V and VII.
  • The location of thrombomodulin on the endothelial surface is important. When the endothelium is intact, the thrombin-thrombomodulin-protein C interaction will inhibit coagulation on the endothelial lining. When the endothelium is damaged, this anticoagulant mechanism will be absent and clotting can continue unopposed.
  • Thrombin and Anti-thrombin III (ATIII)
  • ATIII is a circulating serine protease inhibitor that can bind and inactivate thrombin, as well as several of the activated clotting factors. It has a central role in the in vivo regulation of hemostasis.
  • ATIII has two critical binding sites – one binds thrombin and other activated clotting factors, and the other binds heparin.
  • Binding heparin to ATIII dramatically increases the efficiency of ATIII binding to thrombin and other clotting factors.
  • Congenital ATIII deficiency leads to dangerous thrombotic events.
  • Causes of acquired ATIII deficiency include liver disease, prolonged heparin administration, nephritic syndrome, DIC, sepsis, preeclampsia, oral contraceptives, and surgery.
  • Tissue Factor Pathway Inhibitor (TFPI)
  • TFPI, generated along with the activation of factor X by the TF-VII complex, acts, in a negative feedback manner, to inhibit the further production of factor Xa.
  • Heparan Sulfate
  • The endothelial surface is coated with a mucopolysaccharide that includes a naturally occurring heparin-like component called heparan sulfate which, like heparin, accelerates the binding of ATIII to thrombin.
  • The location of heparan sulfate at the blood-endothelial surface, where activated factors of the coagulation cascade are being generated, helps to promote the antithrombotic property of the normal endothelial lining.

Summary of the Hemostatic Mechanism

  • Under normal circumstances, the hemostatic mechanism is quiescent with many of the potential participants circulating in an inactive form. Only when the endothelial lining is breached is the hemostatic mechanism set in motion.
  • With collagen and tissue factor exposed, the intertwined processes of platelet-mediated primary hemostasis and factor-mediated coagulation begin and rapidly the vascular injury is sealed by a platelet mass into which are incorporated fibrinogen, thrombin, plasminogen and t-PA.
  • The completion of the coagulation process converts fibrinogen to fibrin and the platelet plug is transformed into a fibrin clot.
  • Simultaneously, several properties of adjacent intact endothelium (elaboration of ADP-ases, prostacyclin, thrombomodulin, t-PA, and heparans) serve to prevent the extension of the clot beyond the site of injury.
  • Within the clot, plasmin, generated from the trapped plasmin and t-PA, begins the process of fibrinolysis. Over time, the entire fibrin clot dissolves, new endothelial cells line the vessel, and blood flow is restored.

Laboratory Tests of Hemostatic Function

  • Tests of platelet function
  • Platelet Count – should be the first test ordered in the evaluation of primary hemostasis
  • Provides platelet numbers but no information regarding their function.
  • Normal – 150,000 to 450,000/mm3
  • Platelet count above 100.000/mm3 and normal platelet function assures normal primary hemostasis
  • Less than 150,000/mm3 is defined as thrombocytopenia
  • When the platelet count is under 20,000/mm3, spontaneous bleeding can occur.
  • Surgery with platelet counts less than 70,000/mm3 may result in severe bleeding.
  • Bleeding Time - the most widely accepted test of platelet function
  • Normal – 2 to 9 minutes
  • Prolonged by poor platelet function or thrombocytopenia. Prolonged bleeding time with a normal platelet count implies a qualitative platelet defect. Specialized testing is required to diagnose specific platelet functional defects.
  • Somewhat dependent on technique employed to do the test, so lacks precision and reproducibility
  • Tests of Coagulation
  • Prothrombin Time (PT)
  • Evaluates the coagulation sequence initiated by TF and leading to the formation of fibrin without the participation of factors VIII or IX (the classical “extrinsic pathway”)
  • Normal is 10 to 12 seconds
  • Prolonged with low levels of factors VII, X, V, prothrombin, and fibrinogen
  • Used to monitor warfarin therapy
  • The INR standardizes reagent differences across labs
  • Activated Partial Thromboelastin Time (aPTT)
  • Measures the time to fibrin strand formation via the classical “intrinsic pathway”
  • Prolonged by low levels of factors VIII, IX, XI, and XII, prekallikrein, kininogen. (all factors except VII and XIII)
  • Used to monitor heparin therapy
  • Thrombin Time
  • Measures ability of thrombin to convert fibrinogen to fibrin
  • Prolonged with low levels of fibrinogen, abnormal fibrinogen, or the presence of circulating anticoagulants, such as heparin
  • Also useful in monitoring LMWH, hirudin, andbivalirudin therapy - the INR and the aPTT may be normal or prolonged, but the TCT will be prolonged if therapeutic levels have been achieved.
  • Activated Clotting Time
  • Measures the “intrinsic pathway”
  • Used to monitor heparin therapy
  • Fibrinogen Level
  • Normal – 160 to 350 mg/dL
  • If less than 100 mg/dL, fibrinogen may be inadequate to produce a clot.
  • Fibrinogen rapidly depleted with DIC.
  • Fibrinogen increased during stress response to surgery and trauma.
  • Tests of Fibrinolysis
  • Fibrin Degradation Products and D-dimers
  • Evaluates in vivo plasmin activity by measuring circulating levels of peptides cleaved from fibrin and fibrinogen by plasmin
  • FDPs elevated in any state of accelerated fibrinolysis – advanced liver disease, cardiopulmonary bypass, DIC
  • High levels of fibrin degradation products interfere with fibrinogen function and platelet plug formation
  • Elevated levels suggest intravascular coagulation with fibrin deposition and secondary fibrinolysis.

Test Results______Possible Diagnoses

Prolonged aPTTvon Willebrand’s Disease

PT – fibrinogen – platelets normalFactor VIII deficiency

Factor IX deficiency

Factor XI deficiency

Prolonged PTWarfarin ingestion

aPTT – fibrinogen - platelets normalEarly vitamin K deficiency

Early liver dysfunction

Factor VII deficiency

Prolonged PT and aPTTOver-warfarinization

Fibrinogen – platelets normalSevere vitamin K deficiency

Over heparinization

Factor X, V, or prothrombin

deficiency

Acquired inhibitors

Prolonged PT and aPTTSevere liver dysfunction

Decreased fibrinogenDysfibrinogenemia/afibrinogen

Normal or low plateletsDIC (including meningococcal sepsis)

  • Thromboelastography
  • PT and aPTT evaluate only the initial phase of clot formation
  • Thromboelastography profiles both the initiation and propagation of clot formation, and produces a more accurate picture of the in vivo coagulation process.
  • Used to evaluate hypocoagulable states, hemophilia, rare coagulation disorders, anticoagulant therapy, coagulopathies secondary to dilution, hypercoagulable states (arterial and venous), and coagulation problems during liver transplantation