Chapter 37

Key events in hemostasis

1) Severed vessel 2) Platelets agglutinate (stick together) 3) Fibrin appears (15-20 s in severe trauma and 22 minutes in minor trauma) 4) Occlusive fibrin clot forms in 3-6 minutes 5) Clot retraction occurs within 20-60 minutes 6) Clot is invaded by fibroblasts and organized into fibrous tissue (1-2 weeks).

Big-picture process of hemostasis (prevention of blood loss)

1) Vascular constriction 2) Formation of a platelet plug 3) Formation of a blood clot 4) Healing of vascular damage + recanalization

Platelets (thrombocytes)

1-4 um discs from fragmentation of megakaryocytes. 150,000-450,000/uL of blood.

Heparin action

Binds and potentiates antithrombin III and works rapidly, used acutely.

Embolus

A thrombus that has broken loose and entered circulation.

Clinical presentation of hemophilias A and B

Bleeding after minor trauma

Blood coagulation tests

Bleeding time (from small cut): Normally 1-6 minutes. Largely reflects platelet function. Clotting time: Invert tube every 30 seconds. Normally 6-10 minutes. Since not reproducible, generally not used.

Antithrombin action of fibrin and antithrombin III

Fibrin fibers bind 85-90% of thrombin and localize it to the clot, helping prevent the spread of thrombin to blood and excessive spread of the clot. Antithrombin III (also called antithrombin-heparin cofactor) combines with the remainder of thrombin and inactivates it over 12-20 minutes.

Two pathways for production of prothrombin activator (PA)

Extrinsic pathway: trauma to vessel wall and adjacent tissues. Intrinsic pathway: trauma to blood or exposure of the blood to collagen. *Both pathways are usually activated at the same time*.

Factor XIII

Fibrin stabilizing factor

Factor XIII use in common pathway

Fibrin-stabilizing factor; converts fibrin to stable fibrin -> clot formation

High-moleular-weight kininogen

Fitzgerald factor; (HMWK)

Prekallikrein

Fletcher factor

Common final pathway for extrinsic and intrinsic pathway

Factor Xa (+ Factor V, Ca2+, platelet phospholipids) -> prothrombin activator -> prothrombin to thrombin -> fibrinogen to fibrin and Factor XII to Factor XIIIa -> Fibrin to stable fibrin -> clot formation

Cases requiring vitamin K injection before surgery

Patients with liver disease (cirrhosis, hepatitis) or biliary disease require an injection of vitamin K 4-8 hours before surgery to prevent excessive bleeding.

Factor XI

Plasma thromboplastin antecedent (PTA), antihemophilic factor C

Factor IX

Plasma thromboplastin component (PTC); Christmas factor; antihemophilic factor B

Clot lysis

Plasminogen is trapped in the clot. Injured tissues release tissue plasminogen activator (tPA). Plasminogen is activated by tPA to plasmin, a protease resembling trypsin.

Overall platelet plug process

Platelets accumulate, adhere and contract, attract more platelets, form a plug, and initiate clotting. Recruitment -> adhesion -> activation -> degranulation

Platelet recruitment -> adhesion -> activation -> degranulation

Platelets have glycoprotein platelet receptor (Gplb) on their surface, which adheres to von Willebrand factor (vWF) on the wall of the capillary. Shape change ensues and platelets release granules (ADP, platelet-activating factor (PAF), and TXA (thromboxane-A)), recruiting other platelets to the site of damage.

Factor V

Proaccelerin; labile factor; Ac-globulin (Ac-G)

Production of vitamin K

Produced in the intestine by bacteria. Also obtained in the diet by leafy greens, cauliflower, etc.

Genetics of hemophilias A and B

Recessive X-linked coagulation disorder, so incidence is much lower in females (females have two X's).

Cell surface glycoproteins of platelet membranes

Repel normal, intact endothelium. Adheres to injured endothelium and exposed collagen (exposed collagen activates platelets).

Ca2+

Required from prothrombin -> thrombin, fibrin monomer -> fibrin fibers.

Organelles/products of platelets

Residual ER and Golgi: synthesize enzymes, prostaglandins, *fibrin-stabilizing factor*, PDGF, and store Ca2+. Mitochondria/enzymes: produce ATP and ADP.

Treatment of thrombi and emboli

tPA (tissue plasminogen activator, converts plasminogen to plasmin, which chews up thrombin), embolectomy

Thrombocytopenia

Results from a low number of platelets, causing bleeding from small venules or capillaries. Petechiae are small (< 3mm) blood spots, while thrombocytopenic purpura causes larger spots. Cause is often idiopathic <50,000 platelets/uL -> modest bleeding <10,000 platelets/uL -> life-threatening

Plasma vs. serum

Serum is plasma without fibrinogen.

Factor VII

Serum prothrombin conversion accelerator (SPCA); proconvertin; stable factor

Why razor cuts bleed heavily

Sharp blades make clean cuts, making it harder to platelets to adhere since they normally join to larger surface areas of ragged cuts. *Prothrombin activator* is diminished (normally released by tissues) since trauma is minimal to tissues.

In vitro anti-coagulation

Siliconized containers prevent contact activation of platelets and Factor XII (intrinsic pathway). Blood clotting can be delayed for about an hour.

Electrolyte concentrations in plasma vs. serum

Similar in both.

Petechiae

Small hemorrhages (<3 mm) that result from low platelet levels. Bleeding gums can also be from this.

Platelet plugs in small vs. large holes

Small holes develop in vessel walls daily and are sealed by platelet plugs rather than blood clots. Larger holes have a loose platelet plug at first, but solidifies when fibrin threads form during blood coagulation.

Extrinsic pathway to initiate blood clotting (damage to tissues) Aww

1. Release of tissue factor (TF)/Factor III, tissue thromboplastin. 2. Factor III activates Factor VII to Factor VIIa, complex of the two catalyzes the conversion of inactive protease Factor X to active protease Factor Xa (w/ Ca2+). 3. Factor Xa combines with TF components and other phospholipids from platelets, forming prothrombin activator complex. Prothrombin is split to form Thrombin (in presence of Ca2+). 4. Thrombin activates Factor V that accelerates the protease activity of Factor Xa; produces a positive feedback effect.

Half life of platelets

8-12 days

International normalized ratio

Accounts for differences in tissue factor used to measure prothrombin time. Manufacturer assigns an ISI (international sensitivity index) to each batch that indicates the activity of the tissue factor with a standardized sample. This allows calculation of the International Normalized Ratio to standardize measurements of prothrombin time. INR = (PTtest/PTnormal)^ISI Normal INR: 0.9-1.3 Therapeutic range: 2.0-3.0 (warfarin patients) INR > 4-5 indicates high probability of bleeding

Membrane phospholipids (tissue factor) of platelet membranes

Activate blood clotting via Extrinsic Pathway.

Formation of platelet plug

Activated by contact with damaged endothelium and collagen fibers. Swell with irradiating pseudopods. Contract and release granules (growth factors, clotting factors (*thrombospondin*). Become sticky: adhere to collagen and plasma protein *von Willebrane factor (vWF)* -> facilitates platelet adhesion. Secrete *ADP and thromboxane A2*-these activate other platelets (positive feedback).

Prothrombin description

Alpha 2-globulin (MW = 68 K); 15 mg/dL in plasma. Vitamin-K-dependent synthesis of prothrombin occurs in the liver. Cleaved by PA into thrombin (MW 33,700).

Thrombus

An abnormal blood clot.

Factor VIII

Antihemophilic factor (AHF); antihemophilic globulin (AHG); antihemophilic factor A

Role of endogenous heparin to decrease clotting in areas with slow blood flow

Basophil cells in the blood release heparin into the plasma. Mast cells concentrated in pericapillary regions of the lung and less so in the liver. The lungs and liver receive embolic clots formed in slow-flowing venous blood; heparin prevents further growth of these clots.

Clot retraction

Begins within 20-60 minutes of damage. Fibrin binds to damaged vessel wall. Platelets bind to multiple fibrin fibers and contract via actin, myosin, and thrombosthenin (actomyosin). *The clot cannot contract without platelets*. Clot tightens, expressing serum (squeezing out), and closes vascular defect.

Intrinsic pathway to initiate blood clotting

Blood trauma causes: 1) activation of Factor XII and (2) release of platelet phospholipids 1. Factor XII is activated to XIIa when it contacts collagen (or wettable surface like glass). Blood trauma also releases platelet phospholipids that contain a lipoprotein called platelet factor III. 2. Factor XIIa acts enzymatically to activate Factor (XI) (requires HMW kininogen and accelerated by prekallikrein). Factor XIa acts enzymatically to activate Factor IX. 3. Factor X is activated in the presence of Ca2+ by actions of Factor IXa, Factor VIIIa, and platelet factor III. 4. Last step same as extrinsic pathway (prothrombin->thrombin)

Location of platelets

Bone marrow, lungs, peripheral blood

Hemophilias A and B pathway impairment

Both impair intrinsic pathway.

Cause of thrombocytopenia

Can be caused by an autoimmune disease following a septic event; antibodies are developed against megakaryocytes. This specifically is treated with steroids.

Calcium chelators (in vitro coagulation)

Citrate, EDTA, and oxalate are used in blood collection and storage.

Blood leaking into tissues

Clot dissolves when substances in the clot are activated.

Von Willebrane factor (vWf)

Clotting protein that binds Factor VIII and platelets in vessel walls. Missing or defective von Willebrand factor causes von Willebrand disease (between 1/50,000 and 1/8500). This disease resembles decreased platelet function and is genetic. Is it less often diagnosed since it is not as severe as hemophilia. Recombinant vWf is used as treatment along with other treatments similar to hemophilia A.

Contractile capabilities of platelets

Contain actin, myosin, and *thrombosthenin (actomyosin)* in their cytoplasm.

Fat soluble vitamins

DEAK: D, E, A, K

Hemophilia B description

Deficiency of factor IX. 15% of cases and about 1/60,000 males.

Hemophilia A description

Deficiency of factor VIII. 85% of hemophilia cases and 1/10,000 males.

Plasma or serum for analyses?

Depends on analyte. Coagulation factors can interfere with some analyses.

Plasmin

Digests fibrin fibers and several other clotting factors. Often results in re-opening repaired small blood vessels.

Elimination of platelets

Eliminated by tissue macrophages in the spleen.

Causes of thrombi and emboli

Endothelial roughening (atherosclerosis->can cause a heart attack) and slow flow (prolonged air travel).

Activation of intrinsic pathway

Exposure of Factor XII and platelets to collagen

Fibrinogen

Factor I

Prothrombin

Factor II

Tissue factor

Factor III; tissue thromboplastin

Calcium

Factor IV

Hemophilia B

Factor IX deficiency

Inherited hypercoagulable conditions

Factor V leiden (most common), prothrombin gene mutation, deficiencies of natural proteins that prevent clotting (antithrombin, protein C and protein S), elevated levels of homocysteine, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), elevated levels of factor VIII and factors IX and XI, abnormal fibrinolytic system (hypoplasminogenemia, dysplasminogenemia, and elevated plasminogen activator inhibitor->PAI-1).

Factor VIII deficiency

Factor VIII (produced in the liver) is normally bound to von WIllebrand factor (produced in endothelial cells and bone marrow cells). Activated and separated from von Willebrand factor in vessel walls in response to injury. Deficiency causes hemophilia A, and is treated with recombinant factor VIII replacement.

Hemophilia A

Factor VIII deficiency

Ket steps of blood clotting (see slide 15)

Generation of prothrombin activator (PA) is the rate-limiting step; PA is a complex of activated substances. PA cleaves prothrombin into thrombin (weak protease). Thrombin cleaves four small peptides from fibrinogen->forms fibrin monomer. Fibrin monomer -> fibrin fibers (spontaneous polymerization in presence of Ca2+) Fibrin fibers are converted into cross-linked fibrin fibers by fibrin stabilizing factor (Factor XIII)

Factor XII

Hageman factor

Clinically useful anticoagulants

Heparin and coumarins (warfarin)

Common causes of excessive bleeding (diseases)

Hepatocellular disease, vitamin K deficiency, hemophilia, low platelet count in plasma (thrombocytopenia).

Coumarins (warfarin)

Inhibits VKOR c1 (normally reactivates vitamin K). Depletion of active vitamin K-> depletion of prothrombin, factors VII, IX, X. Slower acting (days); used chronically. Over-anticoagulation: treat with fresh frozen plasma and vitamin K.

Vitamin K deficiency

Lack of bile production or delivery can cause fat malabsorption and vitamin K deficiency.

Heparin

Limited availability physiologically. Used therapeutically and highly negatively charged. Binds antithrombin III and increases its effectiveness 100-1000 fold. Heparin-antithrombin III removes free thrombin from the blood almost instantly. Also removes factors XIIa, XI a, Xa, and IXa. Produced by basophilic mast cells and basophils particularly abundant in pericapillary regions of the liver and lung. Heparin is destroyed by heparinase (produced by bacteria).

Serum

Liquid part of blood after coagulation, so devoid of clotting factors like fibrinogen.

Plasma

Liquid, cell-free part of blood that has been treated with anti-coagulants.

Fibrinogen description

MW=340 K; 100-700 mg/dL in plasma; low levels in tissue fluid. Synthesized in the liver (acute phase reactant; concentration decreases by 25% in inflammation state).

Prothrombin time

Most used method for blood clot testing. Indicates concentration of prothrombin in blood. Process: Blood sample is immediately oxalated so no prothrombin can change into thrombin (lack of Ca2+). Excess Ca2+ and tissue factor is added to oxalated blood. Tissue factor activates prothrombin to thrombin via extrinsic pathway. Time to clot is prothrombin time (normally around 12 seconds). Relationship between prothrombin concentration and prothrombin time is plotted for each lab. Different batches of tissue factor have different levels of activity (since it is obtained from placenta).

Vascular constriction during hemostasis

Myogenic contraction from direct damage to vascular wall (spasm lasts minutes to hours). Local autacoid factors are released from damaged tissues and platelets. Nervous reflexes are initiated from traumatized tissues. Smaller vessels have platelets that release *thromboxane A2*.

Platelet function

No nuclei; can't reproduce. Contractile capabilities (clot retraction) and synthesis properties.

Timeline of extrinsic vs. intrinsic pathway

Occur simultaneously, but the extrinsic pathway can be explosive (clotting in <15 seconds), while intrinsic pathway is slower (1-6 minutes).

Disseminated intravascular coagulation (DIC)

Occurs in the setting of massive tissue damage or sepsis. Dying tissues release tissue factor (component of cell membrane phospholipids) and endotoxins activate clotting factors. Causes wide-spread coagulation in small vessels and manifests as bleeding from multiple sites, as clotting factors are depleted in DIC.

Methods of clotting prevention

Smoothness of endothelial surface, mucopolysaccharide coating (glycocalyx) repels platelets and clotting factors. Thrombomodulin bound to endothelium binds (competes) for thrombin, removing thrombin and slowing the clotting process. Thrombin-thrombomodulin activates a plasma protein, protein C -> inactivates activated Factors Va and VIIIa.

Factor X

Stuart factor; Stuart-Prower factor

Clot extension/enlargement (see slide 17)

Thrombin is bound to platelets and is trapped in the clot. Positive feedback: thrombin 1) acts on prothrombin to generate more thrombin and 2) produces more prothrombin activator by acting on other clotting factors. Additional fibrin monomers and polymers are generated at the periphery of the clot.

Activation of extrinsic pathway

Tissue factor

Formation of fibrin clot after vascular injury

Tissue factor (Factor III; tissue thromboplastin) is released from traumatized tissues (extrinsic clotting pathway) -> leads to activation of PA. Factor III -> active PA Prothrombin -> Thrombin Fibrinogen -> Fibrin -> Cross-linked fibrin Platelet phospholipids from damaged platelet membranes activate multiple stages in blood-clotting process.

Treatment of thrombocytopenia

Treated with platelet infusions effective for 1-4 days each time. Also treated with steroids.

Platelets

Type of clotting factor

Heparin in vitro anti-coagulation

Used in blood collection, heart-lung and kidney machines.

Clotting factors in extrinsic and intrinsic pathways

Used in both pathways. Mostly inactive proteases activated by cascades of both pathways.

Tissue plasminogen activator (tPA)

Used to treat blood clot-related disorders such as myocardial infarction, pulmonary embolism, thrombotic or embolic stroke.

Pulmonary embolus

Usually from deep leg veins. Part of thrombus disengages around 10% of the time. Occludes pulmonary arteries and is potentially fatal. tPA can be life-saving in this case.

Injury to blood and tissues

Usually occurs at the same time; both pathways combine in final common pathway.

Vitamin K role

Vitamin K needed to carboxylate glutamic acid in five important clotting factors: Prothrombin, Factor VII, Factor IX, Factor X, and protein C. This process oxidizes and inactivates vitamin K. Vitamin K epoxide reductase complex 1 (VKOR c1) reduces vitamin K and reactivates it.

Myogenic contraction

When muscle cells are able to contract and relax without receiving signals from nerves, e.g. heart cells.