Hemostasis Overview

Overview of Hemostasis

• Hemostasis involves the interaction of vasoconstriction, platelet adhesion and aggregation, and the coagulation enzyme activation.
• Hemostasis is achieved through balancing bleeding (hemorrhage) and clotting (thrombosis).

4 Major Components of Hemostasis

• The four major components of hemostasis are:
  • Vascular System/Intima
  • Platelets
  • Blood Coagulation Factors
  • Fibrinolysis

Other Components of Hemostasis

• Complements
• Kinin systems
• Serine protease inhibitors

Primary Hemostasis

• Primary hemostasis includes the vascular system (vascular intima) and platelets.
• Vascular injury and desquamation of dying or damaged endothelial cells contribute to primary hemostasis.

Anticoagulant Properties of Intact Vascular Intima

• Intact vascular intima inhibits platelet aggregation and prevents coagulation activation and propagation.
• It enhances fibrinolysis through nitric oxide, a potent vasodilator.
• It controls activation of the extrinsic coagulation pathway using tissue factor pathway inhibitor (TFPI).

Procoagulant Properties of Damaged Vascular Intima

• Damaged vascular intima induces vasoconstriction to seal the wound or reduce blood flow in the area.
• Smooth muscle cells (arteries and arterioles) are involved.
• Collagen binds and activates platelets.
• Von Willebrand factor, stored in Weibel-Palade bodies, facilitates platelet adhesion to exposed subendothelial collagen.
• Adhesion molecules like P-selectin, ICAMs, and PECAMs mediate platelet and leukocyte binding.

Fibrinolytic Properties of Vascular Intima

• Vascular Intima involves removal of fibrin to restore vessel patency and secretes:
  • Tissue plasminogen activator (TPA)
  • Plasminogen activator inhibitor 1 (PAI-1)
  • Thrombin-activatable fibrinolysis inhibitor (TAFI).

Platelet Adhesion

• Platelets roll and adhere to non-platelet surfaces.
• Von Willebrand Factor is required in arteries and arterioles.
• Receptor: GP Ib/IX/V

Platelet Aggregation

• Platelets bind to one another and get activated; allows binding of fibrinogen.
• Fibrinogen that has a receptor: GP IIb/IIIa, and uses ionized calcium.
• Ionized Calcium is necessary for aggregation, in the presence of fibrinogen and GP IIb/IIIa, for the platelet's conformational changes.

Platelet Secretion

• Platelets discharge the contents of their granules.
• Occurs during aggregation.

Platelet Granule Contents

• Platelet a-Granules contain: B-thromboglobulin, Factor V, Factor XI, protein S, Fibrinogen, vWF, Platelet Factor 4 and Platelet-Derived growth factor.
• Platelet Dense Granules (Dense Bodies) contain Adenosine diphosphate. Adenosine triphosphate, Calcium and Serotonin (vasoconstrictor).

Bernard-Soulier Syndrome

• Deficiency or lack of GP Ib/IX/V.

Glanzmann Thrombasthenia

• Deficiency or lack of GP IIb/IIIa.

Secondary Hemostasis

• Activation of a series of coagulation proteins in plasma.
  • Procoagulants
    • Serine Proteases
    • Cofactors
    • Transglutaminase (Factor XIII)

Serine Protease

• Serine Protease is an enzyme that cleaves peptide bonds in proteins and have a serine residue in their active site.
  • Responsible for the proteolytic activation of other coagulation factors like thrombin (Factor IIa), Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, Pre-K.

Procoagulants (Coagulation Factors)

• Catalyzes the cross-linking of fibrin molecules, stabilizing the fibrin mesh formed i.e. Factor XII
• Proteins enhance the activity of serine proteases but do not have enzymatic activity.
• assist in the activation or function of serine proteases. example: Tissue Factor (Factor III), Factor V, Factor VIII, HMWK.
• Fibrinogen as the ultimate substrate of the coagulation pathway.

Fibrinogen Group

• Consumed during the process of coagulation.
• Factors: I, V, VIII, XIII
• Is only found in plasma.

Prothrombin Group

• Consist of vitamin K-dependent factors and come from dietary sources.
• Vitamin K-dependent factors are produced in intestinal bacteria.
• Factors: II, VII , IX , X
• Inhibited by warfarin.
• Stable and well-preserved in stored plasma.
• Is only found in serum.

Contact Group

• Involved in the intrinsic coagulation pathway, Factors: XI, XII, Pre-Kallikrein (Pre-K), HMWK.
• Moderately stable
• Not consumed during coagulation
• Is found in both plasma, and serum.

Intrinsic Pathway

• Consists of: Collagen , Pre-K, HMWK, XII, XI, IX, Vinlla.

Extrinsic Pathway

• Consists of: Endothelial tissue damage, Tissue factor, VIIa, VII.

Common Pathway

• Consists of: Xa, Va, II, Thrombin, I, Fibrin, XIIIa

Fibrinolysis

• The final stage of coagulation is a systematic, accelerating hydrolysis of fibrin bound by plasmin.

Proteins of the Fibrinolysis Pathway

• Plasminogen is a plasma serine protease, with a molecular mass of 92,000 Da, a half-life of 24-26 hr and a plasma concentration of 15-21mg/dL that digests fibrin/fibrinogen.
• Tissue plasminogen activator is a serine protease secreted by activated endothelium, with a mass of 68,000 Da, unknown half life and a concentration of 4-7 μg/dL that activates plasminogen.
• Urokinase is a serine protease secreted by the kidney and activates plasminogen, with a molecular mass of 54,000 Da.
• Plasminogen activator inhibitor-1 is secreted by the endothelium inhibits tissue plasminogen activator with a molecular mass of 52,000 Da, with a half life of 1 hour and 14-28mg/dl plasma concentrate.
• α2-Antiplasmin inhibits plasmin a mass of 51,000 Da, with an unknown half life
• Thrombin-activatable fibrinolysis inhibitor suppresses fibrinolysis by removing fibrin binding sites, with a molecular mass of 55,000 Da, with a half life of 8-10 mins.

Components fo the Plasma Fibrinolytic System

• Plasminogen Activators- Endogenous: Tissue-type plasminogen activator (t-PA), Urokinase. Exogenous: Streptokinase, Acyl-plasminogen streptokinase activator complex (APSAC).

• Plasminogen Inhibitors include alpha-2 plasmin inhibitor, tissue plasminogen activator inhibitor

• Circulating TPA is bound to inhibitors such as PAI-I

• TPA initiates fibrinolysis, and releases TPA from endothelial cells that hydrolyzes fibrin-bound plasminogen.

• Plasminogen is converted to plasmin

• Binding of TPA to fibrin protects TPA from PAI-1 inhibition

• Fibrin-bound = TPA > PAI-1

• Plasma = PAI-1 > TPA

Alpha-2 Antiplasmin

• The principle inhibitor of free plasmin and the primary inhibitor of the fibrinolytic system.
• Competitive binding with the lysine residues in fibrin.

Thrombin-activatable Fibrinolysis Inhibitor

• Prevents the binding of TPA and plasminogen to fibrin.
• Blocks plasmin formation

Fibrin Fragment

• Result of cleaving fibrin.
• Series of identifiable fibrin fragments result

D-Dimer

• Composed of 2 D domains from separate fibrin molecules.
• Crosslinked via Factor XIIIa.
• Specific products of cross-linked fibrin digestion only
• Serves as a marker for Thrombosis and Fibrinolysis

Clinical Findings of Bleeding Disorders

• Petechiae: Small, pinpoint, non-blanching red or purple spots on the skin, caused by minor hemorrhage
• Purpura: Larger, non-blanching red or purple spots on the skin, usually greater than 3mm in diameter, due to bleeding under the skin.
• Ecchymosis: A large bruise caused by bleeding under the skin, typically resulting from trauma or injury
• Hematoma: A localized collection of blood outside the blood vessels, typically in liquid form within the tissue.
• Epistaxis: Nosebleed; bleeding from the nose
• Hemarthrosis: Bleeding into a joint space, often causing swelling and pain
• Hematemesis: Vomiting of blood, indicating bleeding in the upper gastrointestinal tract
• Hematuria: Presence of blood in the urine, which can be a sign of various conditions affecting the urinary system
• Hemoglobinuria: Presence of hemoglobin in the urine, usually due to the breakdown of red blood cells
• Melena: Black, tarry stools caused by the presence of digested blood, often from bleeding in the upper GI tract
• Menorrhagia: Excessively heavy or prolonged menstrual bleeding

Quantitative Platelet Disorders:

• Immune Thrombocytopenic Purpura (ITP): Autoantibodies bind to platelets, resulting in a shortened platelet lifespan (Normal Platelet lifespan in the circulation is 7-10 days).
  • Acute ITP: Usually occurs in children (2 to 4 years old), often follows a viral infection, abrupt onset, (+) petechiae.
  • Chronic ITP: Adult-onset (F > M), insidious, and lasts for 6 to 12 months

Thrombotic Thrombocytopenic Purpura (TTP)

• Characterized by abnormally large vWf multimers in circulation causing platelet agglutination.
• Excessive clotting with low platelet counts.
• Levels of ADAMTS-13 is reduced.
• ADAMTS-13 is a disintegrin and metalloproteinase with thrombospondin motifs 13 that regulates vWF size (cleaves vWF).

Mixing Studies

• Used to investigate the cause of an abnormal coagulation test, specifically prolonged prothrombin time (PT) or activated partial thromboplastin time (aPTT).
• Help determine whether the prolonged clotting time is due to a factor deficiency or the presence of an inhibitor Mix reagents for correction with the patient's plasma.

Clotting Pathways

• Intrinsic: Factors VIII, IX, XI, XII. (8, 9, 11, 12)
• Extrinsic: Factor III, Factor VII (3,7)
• Common: Factors I, II, V, X (1,2,5,10)

Clotting Factors: Plasma, and Serum

• Fibrinogen Group: Factor I, V, VIII, XIII (1, 5, 8, 13); ONLY plasma.
• Prothrombin Group: Factor II, VII, IX, X (2, 7, 9, 10;) ONLY serum.
• Contact Group: Factor XI, XII, Pre-Kallikrein, HMWK (11, 12, Pre-K, HMWK) Plasma, and serum.

Clotting Assays - Results if Deficient Factors Present

• Increased PT: I, II, V, VII, X
• Increased aPTT: I, II, V, VII, VIII, IX, X, XI, XII
• Increased TT: I

Clotting Assays - Fresh Factor, Absorbent, Aged Serum, Aged Plasma

• With fresh factor all factors are corrected.
• With absorbent factor only prothrombin are corrected.
• With aged serum fibrinogen/I-related factor are not corrected.
• With aged plasma all factor except #5 and #8 are corrected. PT = 15 seconds APTT = 31 seconds TT = 10 seconds

Interpretation of Mixing Studies

• FP = Corrected
• AdP = Not corrected
• AS = Corrected
• AgP = Corrected

Interpretation of Mixing Studies - WITH: Presense of AI/LA

• FP = Not corrected
• AdP = Not corrected
• AS = Not corrected
• AgP = Not corrected