Hemostasis Ch4 D&P Flashcards
Coagulation is the process that results in the generation of what compound? What is the ultimate job of this compound?
Thrombin. Which converts soluble fibrinogen into fibrin.
What is the compound responsible for dissolution of fibrin via fibrinolysis?
Plasmin.
What are the ANTI thrombotic properties of endothelium (7)?
Prostacyclin/PGI2 release (its a prostaglandin) > Vasodilation and platelet inhibition (via cAMP etc).
NO release > same as prostacyclin, Vasodilation and platelet inhibition.
Thrombomodulin expression > binds and inhibits thrombin and when bound this inhibits Protein C which in concert with Protein S inhibits factors 5&7 (ie it modulates thrombin and converts it from pro to anticoagulant).
Tissue plasminogen activator (tPA) > converts plasminogen to plasmin in the presence of fibrin»_space; fibrinolysis.
Heparin sulfate expression > accelerates antithrombin binding and inactivation of thrombin and factor X.
TFPI (tissue factor pathway inhibitor) > inhibits tissue factor/activated factor VII complex.
EctoADPase release > degrades locally generated ADP, which limits platelet aggregation.
REMEMBER: THe ANT PT (ie ants working out) to remember this)
What are the procoagulant properties of endothelium (4)?
Tissue factor (III) synthesis. TF in combination with factor VIIa and X forms the extrinsic factor X activation complex.
VWF synthesis storage and release, which supports platelet adhesion to subendothelial collagen (synthesized and stored in Weibel-Palade bodies in vascular endothelial cells).
Plasminogen activator inhibitor/PAI-1 synthesis and release which inhibits fibrinolysis.
Endothelial damage > subendothelial exposure and enhanced plt aggregation & reduction of cell membrane associated thrombomodulin and heparan sulfates that inhibit hemostasis.
Remember: PE is better that TV.
What on platelets serve as receptors for activation, adhesion and aggregation?
Transmembrane and peripheral glycoproteins within the phospholipid bilayer.
What are the three types of granules in platelets and what does each contain?
Alpha > reddish, largest and most numerous and have coag and GFs, and proteins involved in platelet adhesion, aggregation and tissue repair. (Ex fibrinogen, factor V, VWF, thrombospondin, Plt factor 4, PDGF).
Dense granules > store adenine nucleotides, calcium, inorganic phosphates, serotonin (SHEMACG).
Lysosomes > acid dependent hydrolases including glycosidases, proteases, lipases.
What is the platelet circulating lifespan?
five to nine days.
Circulating platlet mass may be sequestered where?
Spleen. Epinephrine induced splenic contraction may increase counts. Or they may get sequestered there during congestion. BUT this does not affect platelet production.
What regulates platelet production and how is it regulated?
TPO is the key in critical for all stages of megakaryopoesis. TPO is important for overall hematopoiesis. Its produced constituatively under steady state conditions (at a constant rate and degraded based on how many plts present) but may increase via marrow stroma cells during severe thrombocytopenia.
**NOTE TPO concentration is related to/inversely correlated with the mass of megakaryocytes and platelets and is NOT related to platelet number.
**Inflammatory conditions can enhance TPO production via hepatocytes, mediated by IL-6 > reactive thrombocytosis.
Other CKs involved in megakaryopoiesis and plt production include stromal cell derived factor 1, CXCR4, FGF-4 (interesting this also regs chondrodysplastic dogs), IL3, GM-CSF, stem cell factor and many other CKs.
How do coag factors assemble on the surface of platelets?
Expression of phosphatidylserine.
How does plt adhesion take place?
Plt exposed to subendothelial collagen, vWF > membrane glycoproteins / GPs ie GPIb-IX-V, GP-VI and alphabeta integrins like GPIIb-IIIa are key players.
GPIb-IX-V is key to gettting plts bound to collagen under high shear, it mediates transient arrest pf plts from flowin blood and weak tethering via binding VWF, tethered plts roll along and and encounted collagen fibrils and bind GPVI > activation of integrins, release of ADP > thromboxane formation. alpha2beta1 integrin txformed to high affinity state binds tightly to collagen and allows firm platelet adhesion and spreading. .
How does platelet aggregation take place?
Platelet aggregation is via alpha IIb Beta3 and is txformed to high affinity state and reinforces firm plt adhesion and plt aggregation by binding fibrinogen
How does platelet granule release take place?
Agonist type and concentration dependent > activation of plt phospholipases > Ca and DAG mobilization > synthesis of TXA2 > irreversible plt aggregation and release.
Release of ADP, serotonin, coag factors including fibrinogen, factor V and factor XI further enhancing plt aggregation and thrombus growth.
What is the role of Phosphatidylserine during platelet activation?
Translocation to the outer membrane > facilitates Assembly of coag factor complexes > thrombin generation.
What is the role of plts in inflammation?
Release of vasoactive compounds (serotonin, plt activating factor), production of CKs and interactions with Neuts.
What is the role of plts in tissue repair?
Release of potent mitogens like PDGF, EGF, FGF
Whats an important point about the size of cat platelets?
Its highly variable and they can be very large so on automated counts may be miscounted as RBCs and look falsely low (pseudothrombocytopenia).
Cat platelet clumping is also an issue in some counting methods.
What is one of the most accurate ways to measure platelets on a machine?
Flow cytometry. The other methods are fair to good for most species, but not good with cat platelets.
What breeds of dogs often appear thrombocytopenic and why?
Cavalier King Charles Spaniels due to giant platelets, which are common in this breed, being excluded from the count. Their platelet mass is normal I think.
Greyhounds generally have lower platelet counts than other dogs breeds but this is normal for them (100,000k).
At what platelet count levels does spontaneous petechial to ecchymotic hemorrhage occur?
Until plt counts are below 20,000
Two conditions where you can see thrombocytosis and in which condition is there increased risk of thromboembolic dz?
Counts would be higher than 800,000/uL»_space; Thrombocytosis.
Thrombocytosis associated with inflammation does not increase risk of thrombosis, BUT
Thrombocytosis associated with Myeloproliferative dz may increase risk.
How do you estimate platelets on a blood smear?
8-10/100X oil > or = to 100,000 plts/ul
6-7/100X oil = 100,000/ul
<3-4 plts/100X oil = significant thrombocytopenia OR less than 1 plt/50 RBCs.
Each platelet per 100X oid = 20,000/ul. If there are plt aggregates that usually means plt counts are good.
Platelets with vacuoles, less intense stanining and decreased granularity may mean what?
Platelet activation in vivo like in DIC or FeLV ( or due to poor sample handling, ie in vitro.)
MPV is an indicator of what?
Average size of plts in circulation and inversely proportional to plt number.
Increased MPV with thrombocytopenia suggests what?
Responsive thrombopoiesis. (NOTE reticulated platelets / increased RNA, are also an indicator of this) MPV greater than 12fL indicates normal to increased megakaryocytopoiesis in the bone marrow.
Decreased MPV is most commonly associated with what three things?
Insufficient megakaryocytes, lack of a megakaryocyte response in the marrow and early immune mediated thrombocytopenia.
How is dx of present of antiplatelet antibody made (ie with ITP?)
There are assays but not that accurate; IFA labeling and Flow cytometry can be useful but don’t help differentiate primary from secondary ITP. Dx of primary ITP is made by process of elimination.
What is the gold standard for assessment of platelet fxn and what conditions would this help dx?
Platelet aggregation.
Tests of this are useful to dx hereditary plt fxn defects like canine thrombopathia or Glanzmann thrombasthenia); or acquired disorders of plt fxn secondary to dz or drug administration (uremia, aspirin, NSAIDS etc.)
What does BMBT measure specifically; when is it prolonged(2); how reliable is it?
Primary hemostasis / plt status; BMBT is the most reliable clinical test for in vivo assessment of platelet function.
It will be Prolonged in thrombocytopenia. Used for animmals with NORMAL platelet counts but questionable platelet function. (ie it tests plt function testing, acquired or congenital).
COAG factor deficiencies do not prolong BMBT.
What does the clot retraction test measure and when is it used? In what conditions is it normal / abnormal? see diagram 4.1 pg 116
Platelet function. Just like BMBT, only used whe a known adequate platelet is present. Thrombocytopenia and admin of antiplatelet drugs will result in poor clot retraction. (think of it as clot shrinkage or tightenining, you want a nice small tight clot).
It tests interactions between plt receptors, thrombin, fibrinogen.
Normal in Canine thrombopathia (CalDAG-GEFI disorder, where there is an abnormal signal txduction affecting fibrinogen receptor and dense granule release), & in dogs with VWD (platelets bind VWf exposed on subendothelial collagen via GP1b).
Abnormal in Glanzmann thrombasthenia (which is a defect in GpIIb-IIIa on platelet surface that links platelets together via fibrinogen)
What are two tests for Von Willebrands dz?
Quantitative Elisa
Collagen binding assay Elisa
How can flow cytometry be used to evaluate platelets?
To determine if plts lack major GPs like GPIIb and GP IIIa and if spp sensitive Abs are present.
Assess fxn via detection of fibrinogen binding activated platelets (via CAP 1, an antibody that detects fibrinogen bound to activated canine platelets).
How does plt granule release take place?
Rapid granule release depends on agonist type and concentration. Various types of agonists activate plt phospholipases which result in Calcium and DAG mobilization and synthesis of
**Thromboxane A2 > irreversible plt aggregation and release.
Stuff like serotonin, coag factors, fibrinogen etc relesed from granules enhance further plt activation and thrombus growth.
Key component of plt activation that has to do with thrombin generation?
Phosphatidylserine translocation to the outer membrane which facilitates assembly of coag factor complexes > thrombin generation.
What is von willebrands factor synthesized by? Whats its function(s)?
Synthesized predominantly by megakaryocytes and endothelial cells.
Glycoprotein needed form platelet adhesion to subenodthelial collagen and serves as a carrier for factor VIII-coagulant, it provides stability for factor VIII-coagulant in circulation.
What is VWD?
What spp are affected?
a dz, inherited or acquired, that involves VWF which is a plasma protein that is needed for normal platelet function.
Its common in dogs but rare in cats, horses and cows.
Factor VIII:Coagulant activity is decreased b/c insufficient VWF to stabilize and protect the protein from degradation by proteases, but there still is some FVIII:C activity so APTT is normal.
What are the signs of VWD?
How may it be treated?
Mucosal hemorrhage, prolionged bleeding from wounds, increased cutaneous bruising. A plasma txfusion or cryprecipitate that provides VWF and Factor VIII:C is necessary for treatment.
May also be treated with DDAVP which is vasopressin that causes release of preformed high molecular weight VWF multimers from Weibel_Palade bodies which works for minor surgical procedures in dogs with Type I VWD.
What are the different types of VWD?
Type 1 > Partial quantitative deficiency of VWF, the VWF present is normal just decreased and MOST CASES are this type. Autosomal inheritance. Severity varies.
Type 2 > Qualitative abnormalities of structure and function and its decreased in plasma. Rare. Autosomal recessive. German shorthair pointers and wirehair pointers.
Type 3 > Severe quantitative deficiency, its severe. Reduced FVIII:C but still some present so APTT is normal. Chesapeake Bay Retrievers, Scottish Terriers, Shetland sheepdogs.
Acquired VWD > When there is high shear stress like aortic stenosis etc, = unfolding of VWF and cleavage by ADAMTS13 (disintegrin and MMP necessary to prevent plt adhesion/aggregation) > into smaller multimers of VWF that aren’t fxnl.
What are the 9 intrinsic platelet function disorders (ie defects in plts themselves)?
Chediak Higashi syndrome > abnormal plt, melanocyte and WBC granules.
Glanzmann throbasthenia > defeciency of GPIIb-IIIa integrin on plt surfaces so they can’t bind fibrinogen.
CalDAG-GEFI disorders > canine thrombopathia, abnormal fibrinogen receptor exposure and impaired dense granule release.
Platelet dense granule > ADP decreased, plt count and morphology normal.
Cyclic hematopoiesis > cyclic fluctuations in plts (and neuts, retics), plt reactivity is defective and dense granules are absent.
Leukocyte adhesion deficiency type III (LAD-III) > Kindlin III deficiency or dysfxn, can’t activate beta subunit type integrins.
P2Y12 > plt ADP receptor, associated with plt aggregation, binding and granule release
Scott syndrome > rare, lack of procoagulant expression on plt surface, decreased Annexin 5 binding & lack of Phosphatidylserine exposure.
Macrothrombocytopenia of Cavalier King Charles Spaniles > mutation in beta 1 tubulin genes and altered megakaryocyte proplt formation/release but no bleeding tendancies but may be miss-diagnosed with thrombocytopenia.
Remember CCCGGGLadPSM
What is the defect in Chediak Higashi, hows it inherited and whats the result and which animals are affected?
Autosoma recessive with lack of leukokcyte, melanocyte and plt granulation. Lack dense granules so deficient in ADP, serotonin, divalent cations etc;
diluted hair color and prolonged surfical bleeding. Mink, cattle, foxes, killer whales, mice.
In cattle mink and people linked to lysosomal trafficking regulator LYST gene.
What is abnormal in CalDAG-GEFI plt disorders? Whats it cause and how is it dx’d?
Inherited defect in signal txduction. Abnormal fibrinogen receptor exposure and impaired dense granule release d/t absent or dyxfunctional Calcium diacylglycerol guanine nucleotide exchange factor I, which is a signal txduction protein in the pathway leading to the conformational change in GPIIb-IIIa necessary for plts to bind fibrinogen which links plts together.
Petechaie and mucosal bleeding. Clot retraction test is normal, need plt aggregation testing to dx.
Basset hound, Eskimo, Spitz.
What is cyclic hematopeosis?
Autosomal recessive disorder in grey collies; cyclic fluctuations in neutrophils, reticulcyts nad platelets and melanocytes are also affected. Its a bone marrow stem cell defect that causes neutropenic episodes every 12 days with high mortality due to infection. Plt reactivity to collagen, PAF and thrombin defective. Plt dens granules absent. Clot retraction and plt adhesiveness impaired. Its a mutation in adapter protein gene AP3; has to do with export of proteins to granules.
What is leukocyte adhesion deficiency type III?
Disorder due to Kindlin 3 deficiency or dysfunction. Key for signal transduction and activation of beta subunit type integrins (Beta 1, 2, 3) on hematopoietic cells. Bleeding similar to Glanzmann thrombasthenia, persistent leukocytosis, susceptible to infections.
What are the two acquired platelet qualitative functional disorders?
Hyporesponsive platelets.
Enhanced platelet function.
What are the causes of hyporesponsive platelets?(5)
Drugs (like COX inhibitors, Aspirin > acetylates COX in plts and inhibits thromboxane A2 production; ibuprofen and other NSAIDS reversibly inactivate COX and nearly all COX2 selective NSAIDs have some COX-1 activity so even the COX2’s have some effect on plts..
Beta lactam antibiotics reversibly inhibits plt fxn by binding agonist receptors.
Calcium channel blockers impair plt fxn by preventing Ca mvmt across membranes.
Uremia may impair plt fxn and prolong BMBT.
DIC produce increased plasma conc of FDPs and they competitively inhibit fibrinogen.
Liver dz associate with impaired plt fxn.
Infections agents like FeLV; E. canis; paraproteinemia of plasma cell myeloma; leukemia and myeloproliferative disorders; some snake venoms.
What are some causes of enhanced plt function?
Nephrotic syndrome (multifactorial) EPO administration Infectious agents and parasites (FIP affects plts directly d/t endothelial cell perturbation or inflammation; heartworm dz d/t parasite products, endothelial damage and erythrolysis.)
What are the four basci mechanisms of thrombocytopenia?
Decreased production
increased consumption
sequestration
excessive loss
In what four situations can you get decreased platelet production or failure of plt production?
This is decreased marrow megakaryocytes with concurrent thrombocytopenia:
- Pure megakaryocyte hypoplasia, this is ab’s directed against megas and can be seen with IHMA (Evans)
- Bone marrow pan hypoplasia (pancytopenia, aplastic anemia) of any cause and starts with neutropenia cause they have the shortest half life
- Myelopthisis where marrow filled with abnormal or non marrow cells
- Infectious agents like ehrlichiosis or other rickettsials associated with thrombocytpenia and FeLV, FIV, EIA, BVD (destroys marrow cells and infect stroma, decrease GF production), ASF.
What are causes of myelophthisis (4 causes) (and what is it)?
where marrow is occupied by other cells like in neoplasm (ie leukemia, myeloma) or other non marrow cells like
stromal proliferation in myelofibrosis or
osteoid in ostesclerosis or
disseminated inflammation in osteomyelitis.
Infectious agents that can trash the marrow?(7)
ehrlichiosis or other rickettsials associated with thrombocytpenia and FeLV, FIV, parvo/panleuk, EIA, BVD (destroys marrow cells and infect stroma, decrease GF production), ASF
What are causes of bone marrow pan hypoplasia where marrow is just empty of precursor cells?
Drugs like chemo or griseofulvin or estrogen, chloramphenicol.
Chemicals like trichloroethylene or benzene
Mycotoxins and plants (ie aflatoxin B and Bracken fern)
Ionizing radiation
FeLV or parvo
What are some causes (3)of platelet consumption or destruction in excess of rate of plt production?
Immune mediated ITP
Drug induced ITP
Increased activation and removal pf platelets (DIC, intravascular parasites, infections)
What are the types of immune mediated ITP?
- Primary Immune mediated thrombocytopenia (abs produced agains plt autoantigens like GPIIb-IIIa and GP Ib-IX and megas can also be targeted.
- With secondary its in SLE, neoplasia, infectious dz or drug administration where ag is absorbed onto plts.
- Can have vaccine induced also.
What are the types of drug induced thrombocytopenia?
Drug induced ITP which can be
- drug independent (ie occurs without drug admin and d/t Abs induced against normal platelet surface by initial exposure to drug and persists without presence of drug) or
- drug dependent/secondary drug induced where the drug must be administered to see the ITP.
- drugs can also cause thrombocytoepnia via non immune mechs or non targeting of platelets by causing direct agglutination and sequestration, excessive loss and removal via normal activation and coag mechs.
