BL 02-27-14 08-09am Hemostasis-Approach to Patient - Thienelt

Question 1

First events in formation of clot:

Answer 1

• Platelet Adhesion, Activation, & Aggregation
• Forms platelet plug
• “primary hemostasis”

Question 2

Stabilization of Platelet plug

Answer 2

• stabilized by formation of fibrin network generated through coagulation cascade
• “secondary hemostasis”

Question 3

Key to cessation of bleeding from small vascular injuries…

Answer 3

= optimal numbers & function of platelets

- Disorders of platelet number / function can lead to bleeding from skin, mucous membranes, brain, or other sites

Question 4

Circulating platelet structure

Answer 4

• small anuclear discoid cell ~2-3 microns in diameter
• contain mitochondria (but no nucleus)
• have three kinds of functional granules: dense, alpha, and lysosomal granules.

Question 5

“Life Cycle” of platelets

Answer 5

• arises from megakaryocytes
• maturation time of 4-5 days
• circulating life span of 9-10 days

Question 6

Location of platelets (normal & pathologic states):

Answer 6

• In pts w/normal spleen size, 80% of platelets are circulating & 20% are in the spleen
• In some pathologic states (e.g., hypersplenism), spleen may contain up to 90% of platelets
• Bone marrow reserve of platelets is limited & can be rapidly depleted after sudden platelet loss or destruction.

Question 7

Newly formed platelets

Answer 7

• larger in size

- termed megathrombocytes

Question 8

Contents of dense granules of platelets:

Answer 8

• contain ATP, ADP, serotonin, and calcium

Question 9

Contents of α-granules of platelets

Answer 9

Contains several proteins essential for platelet function, including…

• procoagulant proteins (fibrinogen, factor V, von Willebrand factor, etc)
• platelet-specific factors for platelet activation
• growth factors such as platelet-derived growth factor

Question 10

Contents of Lysosomal granules of Platelets

Answer 10

• contain acid hydrolases

Question 11

Internal structure of Platelets

Answer 11

• extensive system of internal membrane tunnels called surface-connected canalicular system
• cytoplasmic framework of monomers, filaments, & tubules that constitute the cytoskeleton & allow shape change with activation

Question 12

Surface-connected canalicular system of Platelets - PURPOSE

Answer 12

• contents of platelet granules are extruded through this system during platelet aggregation & secretion

Question 13

Platelet Function

Answer 13

Several important roles in hemostasis, including

• ADHESION to vascular subendothelium at sites of injury to begin hemostatic process
• ACTIVATION of intracellular signaling pathways leading to cytoskeletal changes & release of intracellular granules to enhance platelet plug formation
• AGGREGATION to form platelet plug
• SUPPORT of THROMBIN GENERATION by providing phospholipid surface for coagulation cascade to take place

A continuous & dynamic interaction of vessel, platelet, & plasma components

Question 14

Endothelial prevention of coagulation & platelet aggregation

Answer 14

Endothelial cells of intact vessels prevent

• blood coagulation by secretion of a heparin-like molecule & through expression of thrombomodulin (when bound to thrombin, activates protein C and S)
• platelet aggregation by secretion of nitric oxide & prostacyclin, inhibitors of platelet activation

Question 15

Process of Platelet Adhesion

Answer 15

• With vessel injury, subendothelial components are exposed
• Circulating von Willebrand factor (vWF) adheres to this exposed subendothelium
• Under conditions of high shear flow, circulating platelets then contact exposed subendothelium in a rolling fashion & adhere via intrxns btwn glycoprotein Ib (GP1b)on platelet surface & vWF

Question 16

Things leading to firm adherence of platelet to subendothelial surface

Answer 16

• platelet integrin GPIIb-IIIa (αIIbβ3) increases its affinity for vWF with exposure to soluble agonist or adhesive subendothelial matrix proteins –> tighter binding
• GPVI interacts directly w/ collagen in subendothelium
• Numerous ligands in subendothelium (collagen, laminin, fibronectin) also interact w/ β1 integrins on the platelet surface

Question 17

Soluble agonist that increase platelet integrin GPIIb-IIIa (αIIbβ3)‘s affinity for vWF

Answer 17

• thrombin
• ADP
• Epinephrine
• Thromboxan A2

Question 18

Adhesive proteins in subendothelial matrix that increase platelet integrin GPIIb-IIIa (αIIbβ3)‘s affinity for vWF

Answer 18

• Collagen

- vWF

Question 19

Platelet Activation - Overview

Answer 19

1. shape change & spreading
2. granule release
3. intracellular signaling via soluble agonist & G protein coupled platelet membrane receptors
4. Ca mobilization
5. Activation of phoshoplipase A2 –> release of arachidonic acid from phospholipids
6. Arachidonic acid converted to Prostaglandin H2 by COX-1
7. PG H2 converted to Thromboxane A2 by thromboxane synthetase
8. Thromboxane A2 & other agonist released to further amplify platelet activation
9. Phosphatidylserine in membrane switches from inner to outer leaflet
10. Thrombin generation

Question 20

Platelet Activation – Shape change

Answer 20

• With adherence to injured vessel wall, platelets undergo shape change through cytoskeletal activation
• Become more spherical w/ extended pseudopods
• Spread over exposed subendothelium

Question 21

Platelet Activation – granule release

Answer 21

• After shape change, the contents of platelet granules are released

Question 22

Platelet Activation – Intracellular signaling & Ca mobilization

Answer 22

• After shape change & granule release, soluble agonists (thrombin, thromboxane A2, epinephrine, ADP) interact w/ their respective G protein coupled platelet membrane receptors
• Leads to intracellular signaling & Ca mobilization
• Ca activates phospholipase A2, which releases arachidonic acid from phospholipids
• COX-1 then converts arachidonic acid to prostaglandin H2
• PG H2 is converted to thromboxane A2 by thromboxane synthetase
• Thromboxane A2, along with other agonists, is released, acting to further amplify platelet activation

Question 23

Platelet Activation – Membrane affects

Answer 23

• With platelet activation, membrane reorganization also occurs
• Switches phospholipid phosphatidylserine from inner to outer membrane leaflet, making it available to interact w/ clotting factors
• Leads to thrombin generation

Question 24

Platelet Aggregation

Answer 24

• W/ platelet adhesion & w/ binding of soluble agonists to receptors to amplify platelet activation, GPIIb-IIIa is converted to a high-affinity state where it can bind fibrinogen and vWF.
• Binding of the membrane protein talin to GPIIb-IIIa is the last step to mediate the change from a low-affinity to a high-affinity state
• GPIIb-IIIa can then bind fibrinogen –>bridges / laces platelets together into an aggregate
• Thombin generated through activation of coagulation cascade then converts fibrinogen to fibrin to stabilize the platelet plug

Question 25

Adhesion

Answer 25

- Platelets adhere to damaged vessel wall directly via collagen or indirectly via vWF - Slide - Slide

Question 26

Evaluation of platelet function

Answer 26

CBC w/ peripheral blood SMEAR provides platelet count & allows evaluation of platelet size / granularity --- Is this acute or chronic (review old CBCs) Bleeding time (or platelet function analyzer, PFA-100 test) to Dx platelet dysfunction Other possible mechanisms for low platelets (Hx, esp. drugs, Liver/Kidney function, etc.)

Question 27

Bleeding time – how to do & normal time

Answer 27

- Small incision in skin is made using standardized template - Time until cessation of bleeding is measured - Normal bleeding time = <9 minutes

Question 28

Causes of Normal vs. Abnormal Bleeding times

Answer 28

- Hemophiliac w/normal platelet count and normal platelet function will have a normal bleeding time - Platelet count <100,000/uL will lead to prolonged bleeding time - Qualitative platelet disorder will lead to prolonged bleeding time

Question 29

Platelet aggregation studies

Answer 29

- done to evaluate platelet aggregation in response to a set of agonists - agonists including thrombin, ADP, epinephrine, collagen, arachidonic acid, and ristocetin (an antibiotic which causes vWF to bind to GP1b, inducing platelet aggregation)

Question 30

Platelet Disorders - Classified as either…

Answer 30

- qualitative (abnormal function) or quantitative (not enough or too many platelets) - congenital or acquired.

Question 31

Qualitative Platelet Disorders

Answer 31

Disorders of adhesion Disorders of activation Disorders of aggregation Drug effects

Question 32

Disorders of adhesion

Answer 32

Von Willebrand disease (vWD) | Bernard-Soulier syndrome

Question 33

Von Willebrand disease (vWD) – inheritance/acquirement

Answer 33

- most common congenital bleeding disorder | - can also be acquired if antibodies develop against the vWF molecule

Question 34

Von Willebrand disease (vWD) – can be due to...

Answer 34

Can be due to - inadequate amount of vWF - mutations in vWF gene leading to abnormal protein function

Question 35

vWF role/affect

Answer 35

- vWF plays key role in adhesion of platelets to injured vascular endothelium - Lack of vWF leads to abnormal platelet/endothelial interaction, leading to a primary hemostatic bleeding disorder characterized by mucosal and skin bleeding - VWF also serves as carrier protein for factor VIII, so severe deficiencies of vWF can cause functional factor VIII deficiency and thus defects in secondary hemostasis as well

Question 36

Lab tests for diagnosis of vWD

Answer 36

Include - bleeding time or PFA-100 --> abnormally prolonged w/ vWD - factor VIII level - von Willebrand antigen test (measure amount of vW protein) - von Willebrand activity test (aka ristocetin cofactor activity; measures function of von Willebrand protein using donor platelets) - vWF multimer assays (occasionally obtained when evaluating for qualitative defect in vWF function)

Question 37

Treatment of vWD

Answer 37

- Commonly DDAVP (arginine vasopressin) - enhances release of vWF from endothelial stores - effective for treatment of type 1 vWD (partial quantitative deficiency) but not type 2 (qualitative defects) or type 3 (near-complete absence of vWF) Factor replacement used is some situations Should avoid aspirin & other platelet inhibiting agents.

Question 38

Bernard-Soulier syndrome

Answer 38

- rare autosomal recessive disorder - reduced expression of GP1b on platelet surface reduced - leads to defect in platelet adhesion - Platelet aggregation studies only show abnormal aggregation with ristocetin.

Question 39

Disorders of activation

Answer 39

- Storage pool deficiencies | - EX: gray platelets syndrome (α-granules def.)

Question 40

Storage pool deficiency

Answer 40

- Storage pool deficiency can occur, w/ a deficiency of either dense granules or α-granules

Question 41

Gray platelet syndrome

Answer 41

= Deficiency of α-granules is known

Question 42

Disorders from lack of dense granules

Answer 42

- Several syndromes can be associated w/ lack of dense granules - These disorders can also be acquired when platelets pass across abnormal vascular surfaces (such as cardiopulmonary bypass apparatus) leading to partially degranulated platelets - Disorders can also be due to rare defects in signal transduction pathways within the platelets.

Question 43

Disorders of aggregation

Answer 43

Afibrogenemia | Glanzmann thrombasthenia

Question 44

Afibrogenemia

Answer 44

- rare inherited defects in aggregation - leads to both primary (platelet plug formation) & secondary (formation of cross-linked fibrin) hemostatic defects - Pts have platelet-type mucosal & cutaneous bleeding as well as deep muscle hematomas more characteristic of coagulation defects

Question 45

Defect in primary hemostasis in afibrogenemia

Answer 45

- due to lack of fibrinogen for binding to GPIIb-IIIa to allow platelet aggregation

Question 46

Defect in secondary hemostasis in afibrogenemia

Answer 46

- due to lack of fibrinogen for formation of cross-linked fibrin

Question 47

Glanzmann thrombasthenia

Answer 47

- rare autosomal recessive bleeding disorder - caused by absent or defective GPIIb-IIIa - Platelets can adhere but are unable to aggregate in response to normal agonist stimuli - Pts have petechiae & easy bruising

Question 48

Drug effects in platelet disorders

Answer 48

Major classes of drugs which inhibit platelet function include: - COX inhibitors such as Aspirin - NSAIDs such as Ibuprofen - ADP receptor inhibitors such as Ticlopidine (Ticlid) and Clopidogrel (Plavix) - GPIIb-IIIa receptor antagonists such as Abciximab

Question 49

Quantitative Platelet Disorders

Answer 49

``` Thrombocytopenia Immune thrombocytopenic purpura (ITP) Alloimmune thrombocytopenia Drug-induced immune thrombocytopenia Other non-immune-mediated causes of thrombocytopenia ```

Question 50

Thrombocytopenia

Answer 50

- a low platelet count (less than 150,000/uL)

Question 51

Mechanisms of Thrombocytopenia:

Answer 51

- decreased platelet production - increased platelet destruction or consumption - sequestration of platelets in the spleen

Question 52

PLT count & effect

Answer 52

- Normal PLT count: 150,000-400,00 / ul - - 20-50,000 = spontaneous bleeding (increased risk of hemorrhage w/trauma & surgery) - <10-20,000/uL = life-threatening spontaneous hemorrhagse, such as spontaneous intracranial hemorrhage

Question 53

Thrombocytopenia due to decreased platelet production

Answer 53

- primary bone marrow disorders such as aplastic anemia, myelodysplasia, and leukemia - bone marrow invasion by metastatic cancer, myelofibrosis - bone marrow invasion by infections such as tuberculosis - toxins such as chemotherapeutic drugs, chemicals, and exposure to radiation can injure the bone marrow and lead to thrombocytopenia - severe nutritional disorders such as B12 or folate deficiency can affect megakaryopoiesis - rare congenital disorders

Question 54

Immune thrombocytopenic purpura (ITP)

Answer 54

- most common cause of thrombocytopenia - formerly called idiopathic TP - due to increased destruction - two forms of ITP: acute and chronic

Question 55

Immune thrombocytopenic purpura (ITP) – Mechanism of platelet destruction

Answer 55

- Autoantibodies develop against platelet antigens - Leads to their removal by macrophages of reticuloendothelial system of liver & spleen - similar mechanism to that seen with autoimmune hemolytic anemia - two forms of ITP: acute and chronic.

Question 56

Acute ITP

Answer 56

- usually in children or young adults - often preceded by a viral infection - Onset of thrombocytopenia is sudden & can be severe - Present with petechiae & nosebleeds - Recovery is generally w/in 2-6 weeks w/out treatment or after treatment with steroids.

Question 57

Chronic ITP

Answer 57

- more common in adults - often associated with concurrent autoimmune disorders (e.g., SLE or rheumatoid arthritis), lymphoma, or HIV, although most cases remain idiopathic - Spontaneous remissions are infrequent, and most patients require treatment

Question 58

Treatment of Chronic ITP

Answer 58

- most commonly corticosteroids, IVIG, and splenectomy - rituximab (anti-CD20) to deplete B cells - TPO (thrombopoetin mimicker)

Question 59

Effect of steroids in Chronic ITP

Answer 59

- work by dampening proliferation of B cell clone making the autoantibody - effect seen in 7-10 days of starting treatment

Question 60

Effect of IVIG in Chronic ITP

Answer 60

IVIG acts by blocking splenic Fc receptors to prevent their binding to antibody-coated platelets - effect seen in 1-2 days

Question 61

Effect of splenectomy in Chronic ITP

Answer 61

- Splenectomy works by removing site of autoantibody-induced platelet removal - leads to lasting responses in 60-70% of patients

Question 62

Alloimmune thrombocytopenia

Answer 62

- occurs when pt develops Abs to platelet Ags not present on patient’s own platelets - can occur in setting of platelet transfusions - can occur in neonate through passive transfer of maternal IgG alloantibodies across placenta

Question 63

Drug-induced immune thrombocytopenia

Answer 63

- can occur when Ab recognizes a neoepitope created by the binding of a drug to a platelet surface glycoprotein - Heparin-induced thrombocytopenia (HIT) – can be associated w/ development of thromboemboli due to platelet activation

Question 64

Other non-immune-mediated causes of thrombocytopenia include…

Answer 64

Include - DIC - Sepsis - thrombotic thrombocytopenic purpura (TTP) - hemolytic uremic syndrome (HUS) Thrombocytopenia with these disorders is due to increased platelet consumption

Question 65

Thrombotic thrombocytopenic purpura (TTP)

Answer 65

- characterized by fever, renal insufficiency, microangiopathic hemolytic anemia, mental status changes, and thrombocytopenia - occurs when endothelial damage occurs from variety of mechanisms (e.g., infection, immune complexes, HIV, pregnancy, cancer) - leads to abnormal release of unusually large vWF molecules from storage sites, which mediate platelet adhesion & aggregation, forming diffuse platelet plugs in small arterioles

Question 66

Large vWF multimers in TTP

Answer 66

- present b/c of absence of metalloprotease called ADAMTS13 - normally digests vWF into smaller multimers - Congenital form of disease exists, but usually acquired through development of autoantibodies to ADAMTS13

Question 67

Treatment of TTP

Answer 67

- w/ plasmapheresis to remove large vWF multimers & replace the missing ADAMTS13

Question 68

Hemolytic uremic syndrome (HUS) vs. TTP

Answer 68

- similar presentation to TTP - BUT tends to more often be associated with renal failure - AND tends to occur more often in children

Question 69

Hemolytic uremic syndrome (HUS) mechanism & treatment

Answer 69

- due to damage to the endothelial lining, usually by a bacterial toxin - leads to platelet adhesion & activation - leads to microthrombi formation - often self-limited & generally treated w/ supportive care alone

Question 70

Why to evaluate for bleeding disorder?

Answer 70

- Evaluate cause of acute or chronic bleeding | - Determine potential for excess bleeding prior to invasive diagnostic or surgical procedures

Question 71

Patient evaluation for bleeding disorder should include…

Answer 71

- assessment of historical information - physical examination - performance of basic hemostatic screening tests - further testing depending on results of initial assessment

Question 72

Hx in Bleeding Disorder Evaluation

Answer 72

- Detailed Hx of type, frequency, & amount of bleeding - Some pts may consider appropriate amount of bruising / bleeding to be excessive, making evaluation more challenging - Questions to address include: - Does pt display excessive, prolonged, recurrent, or delayed bleeding? - Has pt ever had opportunity to bleed excessively (physical trauma, skin lacerations, surgery)? - Family Hx of significant bleeding?

Question 73

Interpretation of Bleeding types – Brisk Bleeding from obvious trauma

Answer 73

- suggests a local vascular defect

Question 74

Interpretation of Bleeding types –Prolonged / recurrent bleeding

Answer 74

- likely a more generalized hemostatic disorder

Question 75

Interpretation of Bleeding types – Sudden resumption of bleeding from injured site

Answer 75

- raises possibility of excessive fibrinolysis or abnormal crosslinking of fibrin

Question 76

Interpretation of Bleeding types – Multiple site bleeding

Answer 76

- suggests more severe, generalized hemostatic disorder

Question 77

Interpretation of Bleeding types – Mucocutaneous bleeding

Answer 77

- includes bruising, petechiae, epistaxis, menorrhagia, prolonged oozing after tooth extraction, increased bleeding after aspirin intake - indicative of defect in primary hemostasis (platelet disorder or von Willebrand disease)

Question 78

Interpretation of Bleeding types – Soft tissue/joint/deep bleeding

Answer 78

- more consistent with a defect in secondary hemostasis (coagulopathy)

Question 79

Interpretation of Bleeding types—Suspicious Nosebleeds

Answer 79

Suspicious of bleeding disorder if… - Occur every 1-2 months - Last longer than 10 minutes - Involve both nares - Require medical attention or transfusion

Question 80

Findings on physical exam in bleeding disorder evaluation

Answer 80

- may suggest an underlying disorder - petechiae with thrombocytopenia - enlarged spleen & lymph nodes with chronic infections or malignancies - signs of liver disease such as jaundice or edema with liver coagulopathy - musculoskeletal abnormalities & joint disease with hemophilias - Trauma (accidental or non-accidental) should be considered as a cause of multiple / unusual bruises at any age - Large (>2 in diameter) or indurated purpuric lesions should lead to an evaluation for a bleeding problem.

Question 81

Age differences in Bleeding disorders

Answer 81

- Complaints / signs of easy bruising common in children & many elderly people WITHOUT an underlying bleeding disorder - RARE for children < 1 year of age to show bruising - Recurrent, brief nosebleeds are frequently seen in children

Question 82

Basic screening tests when evaluating excessive bleeding can include:

Answer 82

- Platelet count and blood smear to evaluate for thrombocytopenia or other hematologic abnormalities - Bleeding time or platelet function analyzer (PFA-100) to evaluate primary hemostasis - APTT as a screening test for the intrinsic coagulation pathway - PT/INR as a screening test for the extrinsic coagulation pathway - Thrombin clotting time (TCT) to evaluate for fibrinogen defects, the presence of fibrin split products, or heparin effects - Fibrinogen level Each of these tests may be the single abnormal test in bleeding disorders present in an outpatient.