Coagulation Flashcards
What does vWF bind to on the platelets?
Glycoprotein Ib/V/IX
(“1B59”)
Damanged endothelium–>circulating vWF in plasma attaches to endothelium, platelets attach to vWF using GPIb/V/IX
HIGH YIELD!
Platelets have alpha granules and delta (dense) granules. What do they contain?
*hint* dense only have 4 things whereas alpha has a lot so easiest to memorize contents of dense granule
Dense Granule:
- ADP dense granule which causes vasoconstriction
- ATP
- Serotonin
- Ca++
Alpha Granule:
- thromboglobulin
- P-selectin
- PDGF
- PF4
- platelet fibrinogen
- thrombospondin
- VWF
After vWF attaches to endothelium and platelets, platelets release their granules and change shape with expose their fibrinogen receptors. What receptor binds fibrinogen? What drugs inhibit this receptor?
Glycoprotein IIb/IIIa
GPIIbIIIa inhibitors: Abciximab-Reopro, Integrilin,
Aggrastat
Where is vWF made (two sites)?
Endothelial cells (Weibel-Palade bodies) and megakaryocytes (alpha granules)
Coagulation cascade is divided into intrinsic and extrinsic pathways based on in vitro methods. What activates these pathways?
- Intrinsic path activated by blood in contact with negative charged glass
- Extrinsic path activated by tissue factor (subendothelium)
What are the steps of fibrinolysis (breaking down clot)?
Tissue plasminogen activator (TPA) cleaves plasminogen to plasmin
Plasmin cleaves fibrin into fibrin split products
What are the there inhibitors of fibrinolysis?
- Alpha two antiplasmin inhibits plasmin
- PAI inhibits plasminogen
- TAFI (thrombin activatable fibrinolysis inhibitor)
inhibits binding of plasminogen and TPA to fibrin
How does protein C function?
Thrombomodulin binds to thrombin to activate protein C; with its carrier, protein S, activated protein C inhibits FV and FVIII
How does antithrombin function?
Liver-produced AT binds to heparin and inhibits conversion of II to IIa; also inhibits Xa action
Technically, how does the PTT test work?
• Method
– Take negatively charged surface phospholipid (partial thromboplastin)
– Add PPP + calcium chloride
– Measure the time to clot
- Normally < 40 seconds
- Measures intrinsic & common pathway
- Measures all factors except 7 and 13
Technically, how does the PT test work?
• Method
– Take tissue factor and phospholipids
– Add citrated patient’s platelet-poor plasma (PPP) & calcium chloride
– Measure the time to clot
• Normally < 15 seconds
• Measures the extrinsic and common pathways
Technically, how does the thrombin time work?
• Method
– Take exogenous thrombin
– Add PPP
– Measure time to clot
- Measures common pathway
- Measures conversion of fibrinogen to fibrin
• Doesn’t need Ca or phospholipid
• increased paraprotein, amyloid, heparin, dysfibrinogenemia
Technically, how does the reptilase time work?
• Method
– Take Bothrops atrox venom
– Add PPP
– Measure time to clot
• increased reptilase time in dysfibrinogenemia
• If patient is on heparin, they will have an increased TT but nl reptilase time
• Reptilase measures conversion of fibrinogen to fibrin, but is
insensitive to heparin
• heparinase + protamine will correct TT that is increased in a patient on heparin
Which factor inhibitor will correct with an immediate mixing study but then prolongs after 1-2 hours (time dependent prolongation)?
Factor VIII inhibitor
What happens in mixing studies with:
1) dysfibrinogenemia
2) hypofibrinogenemia
1) Partially corrects
2) Completely corrects
What disorder is characterized by a defect in GPIb/V/IX (CD42), shows LARGE platelets with pseudonucleolus, has impaired ristocetin aggregation?
Bernard Soulier
What does Bernard Soulier show on flow cytometry?
Decreased CD42a, CD42b and CD42d
(which corresponds to GPIb, V, and IX)
What disorder is characterized by a platelet aggregation problem because platelets can’t bind fibrinogen and this is from abnormal GP IIb-IIIa receptor?
Glanzmann thrombasthenia
What platelet disorder shows impaired ristocetin aggregation on PFA?
Bernard Soulier
What platelet disorder shows impaired aggregation with ADP, collagen and epinephrine but NORMAL ristocetin?
Glanzman thrombasthenia
What are the flow cytometry findings in Glanzmann thrombasthenia?
Decreased CD41 and CD61
(corresponds to GPIIb and GPIIIa respectively)
What is the main differential with Glanzmann thrombasthenia and how do you tell them apart?
Glanzmann and Afibrinogenemia
*Glanzmann’s has normal PT and PTT
Afibrinoginemia has increased PT and PTT
What disorder is has giant platelets AND WBC inclusion, is autosomal dominant mutation in myosin heavy chain 9 gene, has thrombocytopenia but very little bleeding and normal PMN function?
May Hegglin Anomaly
What is the mutation in May Hegglin anomaly?
Myosin heavy chain 9 gene
HIGH YIELD!
What disorders can cause giant platelets?
- ITP, May Hegglin, gray platelet syndrome, Bernard Soulier, Montreal plt syndrome, DiGeorge syndrome
- Mediterranean macrothromocytosis
- Sebastian, Fechner, Epstein, and Alport syndromes
What platelet disorder is characterized by decreased platelet aggregation due to deficiencies in either dense granules/alpha granules or both?
Storage Pool Deficiency
normal morphology, no granules on EM
This platelet aggregation suggests what disorder?
Storage Pool Deficiency
NO 2nd wave wtih ADP and Epinephrine
DECREASED collagen and AA
NORMAL ristocetin
INCREASED ATP:ADP ratio
What disorder is characterized by no alpha granules, large gray platelets with no granules, results from cardiopulmonary bypass and platelet aggregation studies are blunted with everything except ADP and epinephrine?
Gray Platelet syndrome
What is the dysfunction in Quebec platelet disorder?
No alpha granules (similar to gray platelet syndrome)
What disorder is X linked, NO DELTA GRANULES on EM, platlets are small and granulated and is characterized by thrombocytopenia, infection, eczema and increased risk of malignancy?
Wiscott Aldrich syndrome
(Chediak Higashi and Hermansky Pudlak also no delta granules)
Which three platelet disorders are characterized by no delta granules on EM?
Chediak Higashi, Wiscott Aldrich and Hermansky Pudlak
What platelet disorder is characterized by no delta granules on EM, increased pigment in reticuloendothelial cells, has swiss cheese platelets, patients have increased AK, nevi, tumors and pulmonary fibrosis and granulomatous colitis and increased vWF?
Hermansky-Pudlak Syndrome
(Chediak Higashi and Wiscott Aldrich are also from no delta granules)
*Affected genes: HPS1 (10q23), HPS3, HPS4, HPS5, HPS6, AP3B1, DTNBP1
What will aspirin use show on platelet aggregation studies?
DECREASED second wave to ADP and epinephrine
ABSENT response to collagen and arachidonic acid
NORMAL ristocetin
What coagulation factor is decreased in patients with amyloidosis?
Factor X
What blood group has decreased vWF?
Blood Group O
vWF increased in exercise, inflammation, pregnancy and estrogen therapy
Newborns have high vWF activity; disease may not be detectable until 1 year of age
What diseases can give you secondary vWF deficiency?
Wilms tumor
Congenital heart disease
Hypothyroidism
What vWF test is very specific and the most sensitive test and it measures vWF activity?
vWF:Rcof
What type of vWD is characterized by low quantity, normal multimers, functionally normal, it is the most common type, sometimes all tests are normal, and is the only type treated w DDAVP?
Type I
Multimer analysis looks like normal, just every band is slightly decreased
What type of vWD is characterized by: decreased large and medium sized multimers and has a disproportionately low vWF:RCo (quality) relative to vWFAg (quantity)?
Type 2a
Multimer analysis shows decreased LARGE AND MEDIUM multimers (top)
What type of vWD is characterized by: mutation in exon 28 of VWF that causes increased affinity of VWF for GP1bα, decreased high molecular weight (HMW) multimers, leads to increase clearance of vWF, increased RIPA, No DDAVP?
To test: mutations in exon 28 of VWF gene
Type 2b
HIGH YIELD!
Multimer analysis: decreased high molecular weight
Must be distinguished from platelet type vWD where there is gain of function in GPIb receptor (here gain of function in vWF but result is same). The enhanced binding in both disorders causing increased clearance of platelets and high molecular weight multimers from the blood thus thrombocytopenia and decreased HMW multimers are characteristic in both disorders
What type of vWD is characterized by: only one to be autosomal recessive (others are AD), severe marked deficiency, absence of vWF, F8 also low, may have nl coag parameters, all multimers low and no DDAVP?
Type 3
Absent multimers
Results from frameshift mutations, nonsense mutations and deletions of the vWF gene
What type of vWD is characterized by: defect in GPIb binding (loss of function of GPIb receptor), but no loss of HMW multimers, vWF made but doesn’t work, decreased vWF:Rco, sometimes nl—RIPA, vWF Ag, and F8 and you should suspect when vwf:Rcof < vwf Ag?
Type 2M
What type of vWD is characterized by: defective binding of vWF to FVIII, hemophilia-like (but AR), women with low F8 think of this dz?
Tests: decreased FVIII, decreased vWFAg:C
levels, normal RIPA, nl multimers, nl ristocetin, normal sequence analysis of F8 gene to rule out hemophilia
Type 2N (Normandy)
On boards if you are given stem that sounds just like a hemophiliac but it is in a woman that has low factor VIII, pick this!
What is the order of frequency for the vWD?
1>2A>2N>2M>2B
What type of vWD is characterized by: mutations in GP1ba resulting in increased binding of GPIba to HMW VWF multimers, the abnormality is on the patients platelets (GP1b) not with vWF, plts bind large VWF multimers, VW multimer test shows decreased large vWF multimers, abn RIPA studies, platelet agg studies show low dose ristocetin aggregation, aggregation with cryoprecipitate?
Pseudo vWD (platelet type)
*mutations in GP1bα gene, whereas VWF 2B has mutations in exon 28 of VWF gene
What disorder is shown in this platelet aggregation study (no response to anything EXCEPT ristocetin and this is an incomplete response)?
Glanzmann’s thrombasthenia or afibrinogenemia
*Remember, platelet agglutination with ristocetin occurs independently of fibrinogen and only partial agglutination is seen with ristocetin emphasizing that for aggregation to occur, binding of fibrinogen to the GpIIb/IIIa receptor is necessary
Which inherited platelet disorder hast he classical finding of “large peroxidase positive granules” in neutrophils?
Chediak Higashi
MUST KNOW COAGULATION CASCADE:
Explain the extrinsic pathway.
The main role of the tissue factor pathway is to generate a “thrombin burst”, a process by which thrombin, the most important constituent of the coagulation cascade in terms of its feedback activation roles, is released very rapidly. FVIIa circulates in a higher amount than any other activated coagulation factor. The process includes the following steps:
Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on tissue-factor-bearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVIIa).
TF-FVIIa activates FIX and FX.
FVII is itself activated by thrombin, FXIa, FXII and FXa.
The activation of FX (to form FXa) by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI).
FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin.
Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which forms a complex with FIX), and activates and releases FVIII from being bound to vWF.
FVIIIa is the co-factor of FIXa, and together they form the “tenase” complex, which activates FX; and so the cycle continues. (“Tenase” is a contraction of “ten” and the suffix “-ase” used for enzymes.)
Explain the intrinsic pathway.
The contact activation pathway begins with formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK),prekallikrein, and FXII (Hageman factor). Prekallikrein is converted to kallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa. Factor XIa activates FIX, which with its co-factor FVIIIa form the tenase complex, which activates FX to FXa. The minor role that the contact activation pathway has in initiating clot formation can be illustrated by the fact that patients with severe deficiencies of FXII, HMWK, and prekallikrein do not have a bleeding disorder. Despite this, interference with the pathway may confer protection against thrombosis without a significant bleeding risk.
Explain the common pathway.
The division of coagulation in two pathways is mainly artificial, it originates from laboratory tests in which clotting times were measured after the clotting was initiated by glass (intrinsic pathway) or by thromboplastin (a mix of tissue factor and phospholipids). In fact thrombin is present from the very beginning, already when platelets are making the plug. Thrombin has a large array of functions, not only the conversion of fibrinogen to fibrin, the building block of a hemostatic plug. In addition, it is the most important platelet activator and on top of that it activates Factors VIII and V and their inhibitor protein C (in the presence of thrombomodulin), and it activates Factor XIII, which forms covalent bonds that crosslink the fibrin polymers that form from activated monomers.
Following activation by the contact factor or tissue factor pathways, the coagulation cascade is maintained in a prothrombotic state by the continued activation of FVIII and FIX to form the tenase complex, until it is down-regulated by the anticoagulant pathways.
What are the regulators of the coagulation cascade?
Five mechanisms keep platelet activation and the coagulation cascade in check. Abnormalities can lead to an increased tendency toward thrombosis:
Protein C is a major physiological anticoagulant. It is a vitamin K-dependent serine protease enzyme that is activated by thrombin into activated protein C (APC). Protein C is activated in a sequence that starts with Protein C and thrombin binding to a cell surface protein thrombomodulin. Thrombomodulin binds these proteins in such a way that it activates Protein C. The activated form, along with protein S and a phospholipid as cofactors, degrades FVa and FVIIIa. Quantitative or qualitative deficiency of either (protein C or protein S) may lead to thrombophilia (a tendency to develop thrombosis). Impaired action of Protein C (activated Protein C resistance), for example by having the “Leiden” variant of Factor V or high levels of FVIII also may lead to a thrombotic tendency.
Antithrombin is a serine protease inhibitor (serpin) that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa. It is constantly active, but its adhesion to these factors is increased by the presence of heparan sulfate. Quantitative or qualitative deficiency of antithrombin (inborn or acquired, e.g., in proteinuria) leads to thrombophilia.
Tissue factor pathway inhibitor (TFPI) limits the action of tissue factor (TF). It also inhibits excessive TF-mediated activation of FVII and FX.
Plasmin is generated by proteolytic cleavage of plasminogen, a plasma protein synthesized in the liver. This cleavage is catalyzed by tissue plasminogen activator (t-PA), which is synthesized and secreted by endothelium. Plasmin proteolytically cleaves fibrin into fibrin degradation products that inhibit excessive fibrin formation.
Prostacyclin (PGI2) is released by endothelium and activates platelet Gs protein-linked receptors. This, in turn, activates adenylyl cyclase, which synthesizes cAMP. cAMP inhibits platelet activation by decreasing cytosolic levels of calcium and, by doing so, inhibits the release of granules that would lead to activation of additional platelets and the coagulation cascade.
