ASCP BLEEDING D/O

Causes of thrombocytopenia (due to platelet destruction)

Immune

(1) Immune thrombocytopenic purpura, acute and chronic

(2) Post transfusion purpura- rare, due to incompatible platelet antigens on transfused plts

(3) Neonatal alloimmune thrombocytopenic purpura- similar to Hemolytic disease of the newborn (as in red cells)

(4) Drug-induced purpura- most common= HIT

Non-immune

(1) Disseminated intravascular coagulation (DIC)- plts lost in large clots

(2) Thrombotic thrombocytopenic purpura (TTP)- vWF multimers are too big, land on plts and cause clumping; plts lost in small clumps

(3) Drugs

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Platelet aggregation studies- uses which agonists, and how do they work

(a) Collagen- strong potency

(b) Arachidonate (arachidonic acid)- strong potency

(c) ADP- moderate to weak, depending on the dose

(d) Epinephrine- weak potency

(e) Ristocetin- strong to weak, depending on the dose

Normal plt agg (A= Epi, B,C,D= collagen, arachidonate and ADP

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Abnormal plt agg  (top line= Ristocetin; others= collagen, ADP and saline)

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When plt function study is abnormal (plt agg study), where do you go from there?  What's the differential

Acquired causes (more common)- most commonly due to Drugs (particularly Aspirin, and NSAIDS) or Uremia (bad kidneys)

-also seen with paraproteins, and others

Congenital disorders of plt fxn-

(a) gIIb-IIIa deficiency- can't bind fibrinogen.  gIIb-IIIa- Rec on plt that binds other plts (aggregation) and fibrinogen for crosslinking.  AKA- Glanzmann's thrombocythemia

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(b) gIb-IX-V deficiency- vWF receptor on plts, lack of adhesion to endothelium; AKA Bernard Soulier disease

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Both (a) and (b) are rare disorders

(c) Alpha, Delta or Alpha/Delta granule deficiency- plt granules deficient in number or function; =storage pool diseases

(d) vWF abnormality- plt's lack the vWF receptor, = platelet type vWD

(e) Cyclooxygenase deficiency

(f) Thromboxane synthetase deficiency

(g) Thromboxane receptor deficiency

e,f,g- all del with the inability of the plt to convert arachidonic acid to thromboxane, and are all exceedingly rare.

ASCP BLEEDING D/O

vonWillebrand's disease

role of fVIII-vWF complex (2); types of bleeding episodes in vWD; types of vWD

fVIII travels with vWF thru the blood- and

(1) fVIII activates intrinsic coag cascade, by activation of fIX.  Intrinsic pathway effects PTT and involves fXII, XI, IX, VIII and X

and (2) plts much be coated with vWF to become sticky, otherwise they will pass over the endothelium

Side notes: extrinsic pathway= fVII and III (and X) and effects the PT; and common pathway (all the lowest denominations of paper $)= I (fibrinogen), II (thrombin), V and X, effects both PT and PTT

vonWillebrand diseases:

95% of bleeding episodes from vWD are mild to moderate, typically consist of mucosal bleeding and bleeding after surgery or dental extractions

Type 1: vWF exists as multimers of varying sizes, inlcuding LMW, IMW and HMW.  T1vWD- has normal distribution of multimers, but overall quantitative defect.  Two tests to use for this dx- (1) vWF antigen test, counts the number of multimers and (2) the ristocetin cofactor.  Both these tests will be decreased by about the same amount: 30-40% of normal.  Tx T1vWD with DDAVP- releases vWF from Wiebel-palade bodies of endothelial cells.  If mild, DDAVP can completely correct the disease.

Type 2: unifying fx for all T2vWD- lack of HMW multimers

-Type 2a- decreased HMW multimers (and intermediate as well), in plasma and some plts.  Ristocetin cofactor and vWF antigen test are both very low

-Type 2b- HMW multimers (only) are removed from the blood by normal platelets b/c they are too sticky, lack of HMW multimers in plasma only; again, Ristocetin cofactor and vWF Ag test are very low

[-Type 2M- qualitative defect in vWF function.  Normal levels (vWF Ag test is normal) but low ristocetin cofactor, leads to decreased vWf function

-Type 2N- deficient binding to fVIII, normal vWF Ag test and normal Ristocetin cofactor; but low levels of fVIII.  Historically may have been misdx'd as Hemophilia A, cause have the same presentation, except no family history]

Type 3: Nearly complete absence of all vWF multimers in the plasma, from either markedly reduced synthesis or production of markedly abnormal vWF.  Nearly undetectable levels of both Ristocetin cofactor and vWF Ag.

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Disseminated intravascular coagulation

conditions a/w; lab evaluation

DIC is always a reponse to an underlying mechanism and mortality is dependent on the underlying disease

Conditions a/w DIC: severe infections; complications of pregnancy (amniotic fluid embolism, premature separation of the placenta, septic abortion, retained dead fetus, retained products of conception after delivery); malignancies; massive tissue trauma; hemorrhagic shock, severe liver disease; burns

Lab evaluation of DIC: (a) platelets- low or show decreasing trend; (b) fibrinogen- low or show decreasing trend [*side note- caution with fibrinogen levels- as is an acute phase reactant and may be elevated by the underlying process.  so need to check multiple levels to show the decreasing trend, rather than a single low level indicating DIC]. (c) FDPs/ d-dimer: elevated; (d) PT- increased; PT will decrease first due to comsumption of fVII, but eventually the PTT will also be elevated

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Vitamin K action, factors involved, warfarin therapy indications and monitoring

Vitamin K- is necessary for activation of factors II, VII, IX and X.  In it's active form, Vit K acts to gamma-carboxylate (add an COOH/COO-) to the glutmaic acid residues of the factor protein. [Ca2+ is needed for the step of carboxylation, and is important for coagulation]  Which creates a Vitamin K epoxide (inactive form)--> which is then converted back to an active form by Vitamin K epoxide reductase. 

Coumadin/Warfarin- acts to inhibit the conversion of Vit K epoxide back to it's active form, thus inhibiting coagulation.

In monitoring warfarin therapy- the PT is effected, and INR is monitored (effects the PT), which for proper anticoagulation- should be between 2-3.  takes 3-5 days to become effective, so in patients actively coagulating/thrombosed, need to overlap with heparin/LMWH until therapeutic.

Diet is important!  Pt's must not eat foods high in Vit K (broccoli, leafy green vegetables, chic peas, and more); also caution in pts requiring antibiotics- doses may need to be decreased, as antibiotics will kill the Vit K producing bacteria in the gut and lead to rapid decreased Vit K, accentuating the warfarin activity

In pts with OD/bleeding from too much warfarin- tx with (1) FFP if severe, uncontrollable bleeding; (2) oral Vit K replacement if not emergent, but bleeding; or (3) just skip the next warfarin dose (ie if INR too high).

Primary indications for warfarin therapy- primary or recurrent thrombosis/embolism; anticipatory risk of coagulation- atrial fibrillation or presence of prosthetic valves.

ASCP BLEEDING D/O

Heparin use-

indications, cautions, the main complication-pathogenesis and outcome, mointoring

DO NOT administer heparin with other anti-platelet drugs (ie aspirin) 

Monitor with PTT- adequate anticoagulation is when PTT is 2X the mean of normal (~60-80 sec)

Must be administered IV or subQ, cannot give orally or IM

The half-life of unfractionated heparin is much lower than other anticoagulants, thus it is still used often today, despite the development of LMWH.  The half-life of hep= 60-90 min

The main complication= heparin-induced thrombocytopenia (HIT): onset- 4-15 days after 1st dose, results in 50% (moderate) drop in platelets, and some risk of bleeding.  Thus, platelets must be checked prior to starting, and monitored at least every few days.

Proposed mechanism- platelet factor 4 (PF4) is synthesized and released from platelets; it's job is to bind and neutralize glycosaminoglycans (=what heparin is).  For whatever reason, some people will create an Ab to the heparin:PF4 complex.  Which will bind platelets and activate them (consume them); which also propagates the release of more PF4 and the cycle continues.

So heparin must be stopped.  And DO NOT give pt more plts.  Cannot use unfrac hep OR LMWH in these pts (must use Argatroban)  Monitor with anti-factor Xa. 

If heparin over-dose causes bleeding- can reverse with protamine sulfate

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Factor defciencies:

where factors are synthesized, congenital factor def, what is most common cause of factor def,

The majority of factors are synthesized in the liver (I (fibrinogen), II (prothrombin), V, VII, VIII, IX, X, XI, XII). Exceptions:

-Tissue factor (fIII)- synthesized in most tissues;

-I, V, vWF and XIII may also be made in megakaryocytes; -vWF is also made in endothelial cells

Congenital deficiencies of fac I, II and V are rare; def VII, VIII and IX and pretty common: fVII def is usually only picked up in pts receiving coumadin

-fVIII def= Hemophilia A,

-fIX def= hemophilia B

-fX def is rare

-fXI and XII def's are actually pretty common, but don't come to diagnosis, does not cause bleeding problems.  fXI assay will actually look MUCH worse than pt's symptoms.

-fXIII def is rare 

The most common cause of factor def is actually acquired, and involves multiple factors.  Most common cause= liver disease- all factors are low except VIII.

Others:

-DIC- consumption of fibrinogen, V, VIII and others

-coumadin and low Vit K states- low II, VII, IX and X

-Proteinuria- decreases XI and XII

Causes of low fVII and thus prolonged PT:  liver dz, DIC, coumadin, diet low in Vit K or on antibiotic tx; (inherited fVII def, uncommon)

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Hemophilia A and B, disease prevalence and categorization

Severe: <1% activity

Moderate: 1-5% activity

Mild: >5% (5-50%) activity

Prevalence: Severe= 55% of cases; moderate= 25% of cases; mild= 20%

Cases of severe deficiency- much more frequently also develop inhibitors from the treatment.  So, 15% of all severe pts will also develop inhibitors. 

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Mixing study

Dx of fVIII deficiency and tx

use of fVIIa

Use the mixing study to determine if the prolonged PT or PTT is due to factor deficiency or presence of an inhibitor.  -Mix half pt serum with half normal serum

-If the PT or PTT corrects- then it's likely a factor deficiency, if it doesn't correct- is more likely a inhibitor is present

To dx fVIII deficiency: the PTT must be increased, the fVIII levels must be decreased.  A 1:1 PTT mixing study should show correction immediately after mixing, BUT the correction will fade (back to abnormal PTT) after ~30 min-2hr (a special situation for fVIII)

For treatment purposes- the Bethesda unit assay quantitates the amount of fVIII Ab.  One bethesda unit- drops the fVIII level by 50%, so each consecutive unit further drops the level by 50% (such that 2 bethesda units gives fVIII levels of 25%, ... and 6 bethesda units drops the fVIII level to 1%

In pts with fVIII inhibitor- the treatment is to start immunosuppressive therapy immediately; IF Bethesda unit is ≤10- give human fVIII (will consume the Ab); IF > 10 units, treat the recombinant fVIIa (which is ridiculously expensive)

Conditions in which fVIIa should be used:

-pts with Hemophilia A or B with inhibitors

-proven fVII deficiency

-intracerebral hemorrhage (controversial)

-Uncontrolled hemorrhage following surgery or trauma (controversial)

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The two most common hereditary thrombotic d/o

Other hereditary d/o

The two most common: (1) Activated Protein C resistance = almost always d/t Factor V Leiden mutation (heterozygous or homozygous)

(2) Prothrombin G20210A mutation (heterozygous or homozygous

Other hereditary thrombotic d/o's:

- Protein C deficiency (always heterozygous, fatal in infancy/fetal life if homozygous)

-Protein S deficiency (same as for prot C def)

-Antithrombin deficiency (same as for prot S/C def)

Rare hereditary d/o-

Plasminogen deficiency

Dysfibrinogenemia

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describe thrombin's role in anticoagulation

Prot C/Prot S activity on thrombin

and Resistance to activated Protein C

generally, thrombin, in its activated form (fIIa), is thought of as a part of coagulation (procoagulant)- however, it is also the "off switch".  Activated thrombin will bind thrombomodulin on the endothelium/bv wall--> and with a conformational change--> activates Protein C

Protein C does not work unless is bound to protein S- which is either free or bound to other proteins.  Only the free fraction can bind Protein C

Protein C/Protein S complex then degrades fVa and fVIIIa, stopping the coagulation cascade.

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Resistance to activated Protein C= most commonly- Factor V Leiden mutation- which is a single amino acid change at position 506 (ΔF506).  Which only effects the binding site of Protein C.  Protein C/Prot S cannot bind, and thus, coagulation continues.

-The factor V leiden mutation does NOT effect the procoagulant activity of fV.

Highly prevalent mutation in caucasian population- effects 50% of caucasians with familial thrombophilia, 20% of caucasians with DVTs; and 3-5% of the general population

FV leiden is the most common risk factor for DVT, but risk in arterial thromboses has not been shown.  OCP's increase the risk of DVTs in these pts- ~30-fold increase (and the risk is ~30 per 10,000 per yr)

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G20210A prothrombin mutation

Risk a/w both fV leiden and prothrombin mut

= a G-->A mutation at the 20210 position on the prothrombin gene; a/w elevated prothrombin (factor II) levels

High prevalence: 20% of cauc with familial thrombophilia; 5-10% of pts with thrombosis; 1-3% of general population

In pts with BOTH- factor V leiden and prothrombin mutations- the risk is disproportionately higher than with just one (synergistic effect)

ASCP THROMBOTIC D/O

Protein C deficiency and types

Most effected individuals- develop venous thromboses as teenagers or young adults; almost exclusively heterozygous, as homozygous is fatal in early life

-levels of functional Protein C: 40-65% of normal

2 types:

(1) makes normal protein, but less amount- so both functional and antigenic protein assays are low

(2) makes normal amount, but abnormal protein- so functional assay is low, antigenic assay is normal

*So functional Protein C assay is more important

ASCP THROMBOTIC D/O

Protein S deficiency and types

-most present with venous thrombosis (not age specific)

3 types: 

(1) normal protein in low amounts- low functional and antigenic protein S assay

(2) normal amounts, abnormal protein- low functional, normal antigenic

(3) increased bound Protein S, less free fraction- normal levels Protein S is ~60% bound and 40% free.  in type 3, more Protein S is bound to C4bBP and not available to bind Protein C

ASCP THROMBOTIC D/O

Antithrombin III deficiency-

what factors are able to escape heparin activity

Antithrombin activity- a protease that binds and inhibits/inactivates several coagulation factors including: Xa and most notably IIa (thrombin). (as well as- fIXa, XIa, XIIa, VIIa, kallikrein, plasmin and C1 (complement))

Heparin binds to AT causing a conformational change and increases AT's activity/binding of the factors. AT is a relatively weak inhibitor on its own.  So heparin improves AT action particularly with thrombin.

HOWEVER- fXa (fibrinogen) is a SHORT molecule and heparin has difficulty binding to the AT:fXa complex.  This explains why when monitoring effectiveness of heparin, you need to check fXa levels.

WHEN AT is deficient- will have little response to heparin tx- THUS, when a patient fails to respond to heparin- think AT deficiency

AT Deficiency: predominantly heterozygous with fxnal AT levels 40-75%; typically present with DVT/PE as teenager or young adult

Tx: FFP can be given to increase AT level; and may need to transfuse with FFP prior to heparin tx if needed in emerent situation

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Factors/conditions/tx that decrease Protein C, Protein S or Antithrombin

Clot formation, Liver disease, coumadin, Nephrotic syndrome, DIC, Vit K deficiency and Heparin- all decrease ALL- Prot C, Prot S and AT

High estrogen states: OCP, Estrogen Tx, pregnancy, and any stimulator of estrogen secretion- all decrease FREE Protein S levels, by increased binding.  OCPs and Estrogen tx may also decrease AT.

Acute phase response will artifactually decrease Prot S (free) levels, so keep in mind in labs- if in acute phase reactant category, likely still have functional levels of Prot S, but assay may say different.

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Antiphospholipid antibodies-

three types, proportions

incidence

clinical and laboratory criteria

three types: two are similar (considered the same ab)- anticardiolipin Ab and anti-Beta 2 glycoprotein I

the other is Lupus anticoagulant

25% of pts have anticardiolipin Ab/anti-Beta 2 glycoprotein I

25% of pts have Lupus anticoagulant (which is not necessarily a/w lupus)

50% have both

Incidence of APA: general pop- 3-5%; recent infection- 30%; HIV +- 20-42%; SLE pts- 18-86% (wide range depending on the study)

Clinical criteria: -vascular thrombosis- one or more clinical episodes of thrombosis in any organ/tissue; OR pregnancy complications- a)  one unexplained fetal loss at or after the 10th weeks; b) one premature birth before 34th week; c) 3 unexplained spontaneous abortions before the 10th week

Laboratory criteria: -Moderate to high levels of IgG or IgM anticardiolipin Ab or anti-B2gpI on two or more occasions at least 12 wks apart; OR Lupus anticoagulant Abs detected on 2 or more occasions at least 12 wks apart

*Need at least 1 clinical criteria and 1 laboratory criteria to be dx'd with APA syndrome

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Antiplatelet Agents

-commonly used for:

-qualitative plt defect caused by:

-Aspirin

-NSAIDS

Antiplatelet agents, commonly used for arterial thrombosis

Common ex's: (proph and tx) Aspirin, Clopidogrel (plavix), NSAIDS; gIIbIIIa inhibitors- (for tx only, not prophylaxis)

Acquired plt d/o induced by aspirin: inhibits cycloxygenase--> decreasing production of thromboxane.  Stops the conversion of arachidonate to Prostaglandin H₂-->which would then be coverted to thromboxane A₂.  Effect is irreversible, in it's active form, half-life of ASA is short, but will stay with the platelet until removed from circulation

Clopidogrel (plavix)- inhibits plts by blocking the the ADP receptor

Acquired plt defect induced by NSAIDS: effect the same pathway as aspirin at the same step, but are reversible.  May involve the cycloxygenase enzymes OR other factors in the pathway.

Glycoprotein IIbIIIa inhibitors: reopro, integrillin, aggrastat- block the gpIIbIIIa receptor on plts, blocking the binding of fibrinogen.  They are administered by IV only and are VERY potent plt blockers.

ASCP THROMBOTIC D/O

points of the coag cascade inhibited by coumadin, unfract hep, LMWH, fondaparinux, hirudin and argatroban

Coumadin- blocks f II, VII, IX and X (=vitamin K dependent factors

Unfractionated heparin- (together with cofactor- AT III) destroys f XIIa, XIa, IXa, Xa and IIa- essentially all activated factors, even some VIIa)

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LMWH- degrades Xa and IIa (works better at these sites than unfrac hep, still requires AT III)

- Fondaparinux (arixtra)- effects fXa

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Hirudin and argatroban- effect fIIa (thrombin). Argatroban- a synthetic direct thrombin inhibitor, derived from arginine; is the drug of choice for prophylaxis or treatment of thrombosis in pts with HIT.

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Lepirudin- a manufactured desulfahuridin, derived from S.cerevisiae, a proven safe thrombin inhibitor in pts with HIT