(pt 3) exam #5 - heme II (cls 546)

fibrinolysis + thrombophilia (updated 5/9 for final)

how do we control coagulation?

inhibitors, cofactors, feedback loops, blood flow (vasoconstriction)

where do all the activated cofactors go?

cleared by the liver

coagulation regulatory mechanisms

• Primary regulators

  • AT ; TFPI ; Protein C

• Also regulating coagulation:

  • Thrombomodulin (TM )

  • Endothelial protein C receptor (EPCR)

  • Heparin cofactor II

  • Protein S

antithrombin (AT)

• Plasma proteinase inhibitor (serpin)

• Activation of AT by vessel wall heparan sulfate proteoglycans (HSPGs)

• Activity enhanced 3 to 4 fold by heparan sulfate (glycosaminoglycan found in ECs)

• Most important inhibitor of serine proteases

  • Inactivates thrombin and other enzymes responsible for thrombin generation

indirect thrombin inhibitors (list)

• Heparin (UFH, HMWH)

• Low molecular weight heparins (LMWH)

  • Enoxaparin

  • Dalteparin

  • Tinzaparin

• Fondaparinux

what are the principle regulators/inhibitors of coagulation?

• Tissue factor pathway inhibitor (TFPI)

• Anti-thrombin (AT)

• Protein C pathway (knocks out intrinsic pathway)

tissue factor pathway inhibitor (TFPI) pathway picture

(fibrinolysis) protein C pathway

how is fibrin formed?

thrombin cleaves off FPA and FPB from fibrinogen

what is fibrinolysis?

process of digesting and removing fibrin

how is fibrinolysis activated?

activated in response to cascade activation and fibrin formation

what happens if you have excessive fibrinolysis? inadequate fibrinoylsis?

• excessive = bleeding

• inadequate = excessive clotting

(players of fibrinolysis) components

• Plasminogen (PLG)

• Plasmin (PLN)

(players of fibrinolysis) activators

• Tissue plasminogen activator (tPA)

• Urokinase type plasminogen activator (uPA)

• Factor XII

• Prekallikrein (PK)

(players of fibrinolysis) inhibitors

• Plasminogen activator inhibitor 1 (PAI-1)

• A2--antiplasmin (AP)

• Thrombin activated fibrinolysis inhibitor (TAFI)

• C-1 esterase inhibitor (C-INH)

• α2 – macroglobulin (α2-M)

(players of fibrinolysis) cell surface receptors

• uPA Receptor (uPAR)

• Annexin 2

• Low density lipoprotein receptor like protein (LRP)

fibrinolysis diagram

(fibrinolysis) plasminogen (PLG)

• Circulates as single polypeptide chain

• Synthesized by the liver

• Activated by plasminogen activators (PAs) to plasmin (PLN) by proteolytic cleavage

• Substrates include:

  • Fibrin, fibrinogen, V, VIII, VWF, complement, several hormones, and platelet surface receptors

(fibrinolysis) tissue-type plasminogen activator (tPA)

• Released from ECs in vivo as single chain molecule (sctPA)

  • Does NOT circulate as a zymogen

    • Fully active in single chain form

  • Binds to fibrin surface

• Intracellular stores released when stimulated

  • Thrombin, BK, histamine, exercise, DDVAP, and so on

• Can also bind to EC surface via receptor--annexin

  • Maintains fibrinolytic potential on undamaged vascular surfaces

(fibrinolysis) urinary-type TPA (uPA) / urokinase

• Found in urine and plasma

• Functions mainly in tissue

  • Digests extracellular matrix

    • Enables cell migration

    • Important in wound healing, inflammation, cancer metastasis

• Converted to two chain active form by PLN, XIIa or kallikrein

• Does not need fibrin as cofactor

what are the 2 major plasminogen activators? how else is plasmingoen activated?

tPA, uPA (urinary type plasminogen activator/urokinase)

• also activated by contact factors (XIIa, Xia, kallikrein)

(fibrinolysis) exogenous activators

• Not used in US

  • Streptokinase

    • Derived from beta-hemolytic strep

  • Staphylokinase (SAK)

    • Produced by S aureus

  • Alteplase--tPA--recombinant tPA

(fibrinolysis) plasmin

• Responsible for degradation of fibrin (or fibrinogen)

• Distinct protein fragments produced

  • Fibrin degradation products (FDP)

• Sites of plasmin cleavage

  • Similar in fibrin and fibrinogen

fibrinogen degradation

• Products formed from fibrinogen cleavage

  • Fragment X (trinodular structure)

  • Fragment Y (binodular D + E)

  • Fragments D and E (uninodular)

fibrin degradation

• Products formed from fibrin cleavage

  • Essentially the same as fibrinogen, except:

    • XIIIa-crosslinked fibrin releases

      • D-Dimer

      • Larger molecules of varying composistion

        • DD/E; YD/DY; YY/DXD

systemic effects of plasmin

• If not controlled, plasmin can degrade

  • Fibrinogen V, VIII, and XII

  • Components of kinin and complement systems

fibrinolytic inhibitors

• Prevent systemic proteolysis

• Act at the:

  • PLG activation step

    • Plasminogen activator inhibitors/PAI

  • Directly on plasmin

    • a2-antiplasmin

    • a2-macroglobulin

  • Directly on thrombin

    • Thrombin-activatable fibrinolysis inhibitor (TAFI)

(fibrinolytic inhibitors) plasminogen activator inhibitor (PAI-1)

• Primary physiological inhibitor of tPA and uPA

• Acute-phase reactant

  • Increases during inflammation, stress

• Decreased levels shift hemostatic balance toward hypercoagulability

(fibrinolytic inhibitors) thrombin-activatable fibrinolysis inhibitor (TAFI)

• Activated by the thrombin/thrombomodulin complex

• Inhibits fibrinolysis

  • Downregulates the cofactor functions of fibrin by cleaving lysine residues

  • Interferes temporarily with the interaction of tPA and PLG with fibrin

    • TAFI has short half-life

fibrinolysis inhibitors / activators chart

• extrinsic Xase: inhibited by AT, TFPI

• intrinsic Xase AND prothrombinase: inhibited by AT, APC, a2-macroglobulin

• factor IIa (thrombin): inhibited by TM, AT, HCII

• plasminogen → plasmin: inhibited by PAI-1/2; TAFI; a2-antiplasmin/a2-macroglobulin

what are the phases of cell-based coagulation theory?

• initiation (thrombin spark)

• amplification

• propagation (thrombin burst)

• fibrin formation

• termination (fibrinolysis)

difference in coag cascde and cell based theory

• coag cascade

  • extrinsic/intrinsic pathways work independently & meet at common end point

  • both make large amt of thrombin to form clot

  • coag factors control rate of coagulation, cells = phospholipid surface

• cell based

  • extrinsic pathway initiates process—generates small amt of thrombin @ thrombin spark

  • followed by intrinsic pathway—works on platelets to produce thrombin burst (no role of factor XII)

  • cells control the coagulation

difference between thrombus and clot?

• Clot = forms on the outside of a vessel on tissue (extravascularly)

• Thrombus = forms on the inside of the lumen of a vessel (intravascularly)

what mechanisms are involved in abnormal clot formation? (3)

• lack of inhibitors to clotting

• stimulation of clotting

• problems w activators or inhibitors to fibrinolysis

what is ischemia?

lack of blood flow when a clot blocks a blood vessel

what is necrosis?

cell/tissue death due to blocked vessels

what is an embolism?

clot or thrombus moves from site of creation to another site where it blocks blood flow

what is a thromboembolism (TE)?

thrombus that moves from site of creation to another site where it blocks blood flow

white thrombi

• ARTERIAL THROMBI

• Composed primarily of platelets and fibrin

  • Few leukocytes and erythrocytes

• Usually form at:

  • Regions of disturbed blood flow

  • Sites of damage to endothelium

    • Atherosclerotic plaques

• Plaque

  • Composed of lipids, fibrous connective tissue, macrophages, and smooth muscle cells

how are white thrombi (aterial thrombi) formed?

• created when plaque ruptures

  • Exposing thrombogenic material in subendothelium to blood

  • Activation of platelets and plasma coagulation factors

  • Fibrin formation → thrombus

• Can lead to embolization

  • Myocardial or cerebral infarction

• Therapy

  • Platelet-inhibiting drugs (aspiring, clopidogrel, ticlopidine) or thrombolytic therapy

risk factors for developing ARTERIAL thrombi

• Traditional risk factors:

  • Hypercholesterolemia ; hypertension

  • Smoking ; physical inactivity

  • Obesity ; diabetes

• More recently recognized risk factors

  • Hyperhomocysteinemia, ↑Lp(a), oxLDL

how to detect arterial thrombi?

• Standard hemostasis--not sensitive or specific

• "new" potential tests:

  • Sensitive, still not specific

    • Hyperhomocysteinemia

    • ↑ Elevated Lp(a)

    • ↑ fibrinogen

    • ↑ D-dimer

    • ↑ PAI-1 or ↓ t-PA

    • ↑ high sensitivity CRP (hsCRP)

what is the role of inflammation in clots/thrombi formation?

increases vascular occlusion (often precedes it) increase inflammation increases vascular risk

if arterial thrombi are called white, what are venous thrombi called?

red thrombi

what are venous thrombi composed of?

RBCs

red/venous thrombi

• Composed primarily of RBCs

• Most commonly occur in veins of lower limbs

  • Thrombophlebitis

    • Superficial veins of legs

    • Usually benign

  • Deep vein thrombosis (DVT)

    • Deep veins of legs

    • Distal thrombi--less serious than thrombi is proximal veins (popliteal, femoral, oriliac)

• Venous thrombi lead to serious potential complications

  • Pulmonary embolism (PE)

    • DVT → PE: VTE

how do venous (red) thrombi form?

• Occurs when activation of blood coagulation exceeds the ability of the natural protective mechanisms to prevent fibrin formation

• Laboratory diagnosis difficult

• Objective diagnostic tests--visualization of thrombus

risk factors for VENOUS thrombi

• Venous stasis

• Vessel wall damage

• Factor V Leiden (FVL) and APC resistance

• Deficiency of protease inhibitors (AT, PC, PS, HCII)

• Elevated prothrombin levels (Prothrombin 20210)

• Antiphospholipid antibodies

• Hyperhomocysteinemia

• Decreased fibrinolytic activity

• Malignancy ; surgery

• Miscellaneous

  • Advanced age, obesity, blood type, pregnancy, OC/HRT, smoking, hypertension, hyperlipidemia

**the more risk factors, the most likely the clot forms

microparticles in thrombosis

• Cell-derived products which play key role in thrombus formation

• Released from cell membrane during apoptosis or activation

• Several roles

  • Have procoagulant potential

  • Play role in inflammation, angiogenesis, and immune response

  • Promote anticoagulant functions and fibrinolytic response

• Derived from platelets, ECs, RBCs, WBCs, cancer cells

• Differ in expression of phosphatidyl serine and/or TF

• Controversy--cause of consequence of thrombosis

what makes someone hypercoagulable?

when more procoagulant activity is happening than anticoagulant activity

virchow’s triad

hypercoagulability, stasis, and vessel injury

• made by rudolph virchow to develop/propose mechanism for PE

what are the 2 genes linked to hyperhomocysteinemia? how is it treated?

• genes: cystathionine B synthase (CBS) + methylenetetrahydrofolate reductase (MTHFR)

• treatment: vitamin supplements (folic acid/B6/B12), dietary changes

thrombophilia

any disorder with an increased risk fo VTE ; aka hypercoagulability

hereditary thrombophilia

• Most inherited alterations associated with:

  •  ↑ in procoagulant potential

  • ↓ in natural inhibitors of clotting

  • Abnormalities of fibrinolysis or platelet activation

• Inherited thrombophilic defect

  • Often requires some other (acquired) risk factor—surgery, pregnancy, immobilization, estrogen therapy, obesity, trauma, infection

  • Combination of hereditary predisposition and acquired predisposition

    • Accelerates and exaggerates thrombotic process

    • "Multiple hit" theory of thrombosis

indicators of hereditary thrombophilia

• Usually associated with venous thrombosis

• Suggested by:

  • Venous thromboembolism at a young age (prior to 43)

  • Recurrent venous thromboembolism

  • Family history of venous thromboembolism

  • Thrombosis in an unusual site

    • Cervical or visceral vein

    • Retinal ; cerebral vein

most common inherited thrombophilias (5)

1. Antithrombin (AT or AT3)

2. PC

3. PS

4. Factor V Leiden

5. Prothrombin 20120 mutation

**account for 30% of initial DVT cases ; 50% normal levels assoc w increased risk

(thrombophilia) antithrombin deficiency pathophysiology

• >200 different mutations described

  • Type I deficiency--quantitative deficiency

  • Type II deficiency—qualitative defect

    • Mutation of either heparin binding site or thrombin binding site

indications of hereditary AT3 deficiency

• Incident or recurring VTE

• Family Hx of VTE

• Recurring miscarriage or pregnancy complications

• Hx of arterial thrombosis (<50 yrs old)

• Hx of arterial thrombosis in patients w/ no other risks for atherosclerosis

aquired antithrombin deficiency

• Decreased production—severe liver disease

• Increased consumption

  • Acute thrombosis, DIC

• Increased clearance or loss

  • Nephrotic syndrome ; heparin

• High dose OCs and late pregnancy

  • Increased FIB and coag factor activity (VII and X)

  • Decreased ATIII

laboratory diagnosis of antithrombin defiency

• Functional assays

  • progressive AT assay

    • Quantifies the ability of AT to neutralize thrombin or Xa w/o Heparin

  • heparin cofactor assay

    • Measures the ability of anti-thrombin to bind heparin & neutralize thrombin or factor Xa

• Antigenic assays (nonfunctional)

  • Immunologic--chromogenic

  • Differentiate type I and II (quantitative vs qualitative)

treatment of antithrombin deficiency

• Heparin

• AT concentrates

• Prophylactic therapy usually unnecessary

• If less than <30% AT3 present, may do oral anticoagulants (Coumadin. Eliquis, Pradaxa etc)

(thrombophilia) protein C deficiency pathophysiology

• Reduction of PC to about 50% or normal predisposes to venous thrombosis

• Autosomal dominant inheritance

  • 6% of familial thrombophilia cases

• >180 different mutations described

  • Type I deficiency--quantitative deficiency

  • Type II deficiency--qualitative deficiency

how is protein C activated?

binding of thrombin to thrombomodulin

what does activated protein C (APC) do?

degrades Va and VIIIa

what are the cofactors for protein C?

protein S and Ca2+

how does protein C deficiency work?

• Predisposes to thrombosis because:

  • Decreased capacity to destroy FVa and FVIIIa

  • Results in an increased generation of thrombin and fibrin

  • Loss of cytoprotective effect (anti-inflammatory and anti-apoptotic) of APC binding to EPCR

• Copperhead snake venom activates PC

(thrombophilia) protein S deficiency pathophysiology

• Protein S circulates in 2 forms

  • Inactive form bound to C4b-BP – complement (60%)

  • Unbound or free form (40% )

    • Only free PS has PC cofactor activity

• Absolute and relative amounts of free PS

  • Determined by plasma concentration of C4bBP

• ↓ free PS

  • Prothrombotic tendency due to inadequate APC inactivation of FVa and FVIIIa

• DRW used in Protein S activity testing

(protein S deficiency) presentation, lab evaluation, treatment

• Clinical presentation

  • Similar to that of PC deficiency

  • ~6% of familial thrombophilia cases

• Laboratory evaluation

  • Measure both total and free PS antigen levels

    • Immunologic assays

  • Functional assay based on ability to serve as cofactor for anticoagulant effect of APC

• Treatment—similar to PC deficiency

(thrombophilia) activated protein C resistance pathophysiology

aka Factor V Leiden

• In vitro—inability of activated protein C to prolong clotting tests when added to a test system

  • Due to a diminished ability to destroy FVa

• In vivo—inadequate FVa inactivation

  • Increased production of thrombin and possibly thrombosis

cause of activated protein C resistance / factor V leiden

• 90% of cases of APCR due to FV Leiden

  • Arg→Gln mutation at AA #506

  • Mutant FVa protein resistant to APC inactivation because APC cleavage site is altered

• 10% of patients have alternate mutation in factor V

how does activated protein C work in factor V leiden?

if factor V is mutated, it becomes resistant to the inactivation by APC

• leads to inability to stop coagulation = clot formation

(activated protein C resistance / factor V leiden) laboratory dx & treatment

Laboratory Dx

• Requires both clot based functional assay (APCR) and PCR-based molecular assay for FVL

Treatment

• Same as acute thrombosis

• Prophylactic treatment--only if at increased risk or have recurrent thrombosis

(thrombophilia) prothrombin mutation 20210 pathophysiology

• Glycine → alanine substitution in 3' untranslated region of the prothrombin gene

  • CAUSES INCREASED EXPRESSION OF PROTHROMBIN

• Associated with a mild elevation of plasma prothrombin levels (115%-130%)

• Increased risk of thrombosis due to:

  • Increased prothrombin causing increased thrombin generation

• Accounts for ~18% of familial thrombophilia cases

testing for prothrombin mutation 20120

• Molecular testing for the single point mutation is required

• Other coagulation screen are unreliable

other players in thrombosis risk (5)

• Heparin cofactor II deficiency

  • Inherited, DIC, liver disease

• TFPI variant

  • TFPI gene mutation increased VTE risk

• ABO blood groups

  • Type B and A1 have more VWF & VIII ; type O has less

  • VWF & VIII are acute phase reactants

• Hyperhemocysteinemia (HC)

  • CBS or MTHFR gene mutation

• Fibrinolytic disorders

  • Dysfibrinogen, decreased plasminogen or tPA, or PAI (excess)

list of the major anticoagulants (6)

• unfractionated heparin

• low molecular weight heparin

• warfarin (coumadin)

• dabigatran, bivalirudin (pradaxa, angiomax)

• rivaroxaban, apixaban (xarelto, eliqus)

• fondaprinux (arixtra)

mode of action for unfractionated heparin

inhibits XI, IX, thrombin

• also inhibits Xa

mode of action for low molecular weight heparin

inhibits X & thrombin

mode of action for warfarin/coumadin

targets thrombin, VII, IX, X (2, 7, 9, 10)

• vitamin K antagonist

mode of action for dabigatran, bivalirudin (pradaxa, angiomax)

direct thrombin inhibitor

mode of action for rivaroxaban, apixaban (xarelto, eliquis)

direct Xa inhibitor

mode of action for fondaprinux (arixtra)

indirect Xa inhibitor (binds to AT)

anti-platelet drugs / site of action

• aspirin / thromboxane A2 (COX inhibitor)

• clopidogrel (plavix), prasugrel, ticlopidine / P2Y12 receptor (ADP receptor)

• abciximab, tirofiban, eptifibatide / glycoprotein IIb/IIIa inhibitor

heparin

• Administered parenterally (IV or Sub-Q)

  • Does NOT have direct effect on blood clotting

  • Increases AT's ability to neutralize serine proteases

• Extracted from porcine intestinal mucosa or bovine lung

  • UFH--MW between 5,000 and 30,000 Da

  • LMWH--MW between 4500-5000 Da

    • Prepared by enzymatic/chemical depolymerization of UFH

what test is used to monitor heparin therapy?

PTT/INR

standard/unfractionated heparin (UFH)

• Catalyzes inhibition of most serine proteases

• Influenced by "heparin-binding proteins" (acute-phase reactants) and high FVIII levels

LMWH & fondaparinux

• Produces majority of its effect by catalyzing interaction of AT and FXa

• More reliable pharmacokinetics because

  • Significantly less binding to heparin-binding proteins

• Typically does not require routine laboratory monitoring

  • If monitoring needed (obese patients, renal disease, etc.)—use anti-FXa assay

what anticoagulant is monitored using the PT?

warfarin

(oral anticoagulants) coumadin drugs

• Sodium warfarin or dicoumarol

• Inhibit coagulation by interfering with vitamin K action in the liver

  • Blocks vitamin K-dependent carboxylation of target proteins

  • Resulting in release of nonfunctional molecules in the plasma

  • (γ carboxylated proteins)

• Biologic effect—takes several days to be achieved

  • Half-life of preformed biologically active "normal" clotting factors

  • FVII disappears most rapidly (T½~6 hours)

what anticoagulants act through Anti-Xa activity?

Xarelto, Eliquis, Lixiana, (Rivaroxaban, Apixaban, Edoxaban)

what mechanism does Dabigatran or Pradaxa use to anticoagulate?

thrombin inhibitor

what does Clopidogrel or Plavix do?

platelet inhibitor through the ADP receptor (P2Y12)

how does aspirin inhibit platelet function?

arachidonic acid pathway

• COX2 inhibitor thru TXA (thromboxane A2)

acquired thrombohemorrhagic conditions (list)

• Acquired defects as common as inherited

  • Clinical presentation can range from thrombosis to bleeding--thrombohemorrhagic conditions

• Acquired fibrinolytic defects

• Anti-phospholipid antibodies (aPL)

• Heparin-induced thrombocytopenia (HIT)

  • Flashback to HIT vs HAT

acquired fibrinolytic defects

• 30-40% of patients w thromboembolic disease

  • Have impaired fibrinolytic function

  • Seen post-surgery, coronary disease, OC, third trimester pregnancy, certain infections, drugs, malignancies

  • Increased plasma PAI-1 most common

    • +/- decreased t-PA

  • Long lasting inflammatory response

    • Elevated PAI-1 (acute phase reactant)

antiphospholipid antibody syndrome (APLS)

• Include:

  • Lupus anticoagulant (LA)

  • Anticardiolipin antibodies (aCL)

  • Several subgroups--antibodies to PLs and PL binding proteins

• Produced after certain infections, exposure to medications, in autoimmune disorders

• Most common cause of acquired thrombophilia

  • 5-30% of patients w VTE are positive for LA

antiphospholipid antibody syndrome (APLS) pathophysiology

• Laboratory phenomenon

  • Prolongation of phospholipid-dependent clotting assays in vitro—hence term "anticoagulant"

• Usually no bleeding diathesis

• More often associated with increased risk of arterial and venous thrombosis

major clinical features of APLS

• Venous thromboembolism (VTE)

  • DVT, PE

• Arterial thromboses

  • Stroke, TIA, MI

• Recurrent miscarriages

antibodies in APLS

• Antigenic specificities of "aPL" antibodies

  • Usually IgG, can be IgM

    • Do NOT recognize PLs directly

  • Recognize phospholipid-binding proteins

    • Ex: B2 glycoprotein-I,  prothrombin, V, VII, PC, PS, TFPI

  • aPL antibodies associated with:

    • Autoimmune disorders (SLE, Sjogren syndrome, RA)

    • Infections (malaria, parasitic)

    • Drugs (neuroleptics, quinidine etc)

  • aPL antibodies in the general population (~3-10%)

lab evaluation of APLS

• immunoassays

  • Originally designed to detect ABY that recognize PL components

    • ELISA for anticardiolipin, ABS, anti-PS

  • New assays designed to detect ABS recognizing phospholipid cofactors (ex: anti-Beta2GPI)

• coagulation assays

  • AB reacts w phospholipids in the test system and prolongs clotting

  • Sensitivity of assay influenced by reagent used

  • Could be an incidental finding w/ prolonged coag screen