BL 02-27-14 08-09am Hemostasis-Approach to Patient - Thienelt Flashcards
First events in formation of clot:
- Platelet Adhesion, Activation, & Aggregation
- Forms platelet plug
- “primary hemostasis”
Stabilization of Platelet plug
- stabilized by formation of fibrin network generated through coagulation cascade
- “secondary hemostasis”
Key to cessation of bleeding from small vascular injuries…
= optimal numbers & function of platelets
- Disorders of platelet number / function can lead to bleeding from skin, mucous membranes, brain, or other sites
Circulating platelet structure
- 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.
“Life Cycle” of platelets
- arises from megakaryocytes
- maturation time of 4-5 days
- circulating life span of 9-10 days
Location of platelets (normal & pathologic states):
- 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.
Newly formed platelets
- larger in size
- termed megathrombocytes
Contents of dense granules of platelets:
- contain ATP, ADP, serotonin, and calcium
Contents of α-granules of platelets
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
Contents of Lysosomal granules of Platelets
- contain acid hydrolases
Internal structure of Platelets
- 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
Surface-connected canalicular system of Platelets - PURPOSE
- contents of platelet granules are extruded through this system during platelet aggregation & secretion
Platelet Function
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
Endothelial prevention of coagulation & platelet aggregation
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
Process of Platelet Adhesion
- 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
Things leading to firm adherence of platelet to subendothelial surface
- 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
Soluble agonist that increase platelet integrin GPIIb-IIIa (αIIbβ3)‘s affinity for vWF
- thrombin
- ADP
- Epinephrine
- Thromboxan A2
Adhesive proteins in subendothelial matrix that increase platelet integrin GPIIb-IIIa (αIIbβ3)‘s affinity for vWF
- Collagen
- vWF
Platelet Activation - Overview
- shape change & spreading
- granule release
- intracellular signaling via soluble agonist & G protein coupled platelet membrane receptors
- Ca mobilization
- Activation of phoshoplipase A2 –> release of arachidonic acid from phospholipids
- Arachidonic acid converted to Prostaglandin H2 by COX-1
- PG H2 converted to Thromboxane A2 by thromboxane synthetase
- Thromboxane A2 & other agonist released to further amplify platelet activation
- Phosphatidylserine in membrane switches from inner to outer leaflet
- Thrombin generation
Platelet Activation – Shape change
- With adherence to injured vessel wall, platelets undergo shape change through cytoskeletal activation
- Become more spherical w/ extended pseudopods
- Spread over exposed subendothelium
Platelet Activation – granule release
- After shape change, the contents of platelet granules are released
Platelet Activation – Intracellular signaling & Ca mobilization
- 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
Platelet Activation – Membrane affects
- 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
Platelet Aggregation
- 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
Adhesion
- Platelets adhere to damaged vessel wall directly via collagen or indirectly via vWF
- Slide
- Slide
Evaluation of platelet function
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.)
Bleeding time – how to do & normal time
- Small incision in skin is made using standardized template
- Time until cessation of bleeding is measured
- Normal bleeding time = <9 minutes
Causes of Normal vs. Abnormal Bleeding times
- 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
Platelet aggregation studies
- 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)
Platelet Disorders - Classified as either…
- qualitative (abnormal function) or quantitative (not enough or too many platelets)
- congenital or acquired.
Qualitative Platelet Disorders
Disorders of adhesion
Disorders of activation
Disorders of aggregation
Drug effects
Disorders of adhesion
Von Willebrand disease (vWD)
Bernard-Soulier syndrome
Von Willebrand disease (vWD) – inheritance/acquirement
- most common congenital bleeding disorder
- can also be acquired if antibodies develop against the vWF molecule
Von Willebrand disease (vWD) – can be due to…
Can be due to
- inadequate amount of vWF
- mutations in vWF gene leading to abnormal protein function
vWF role/affect
- 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
Lab tests for diagnosis of vWD
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)
Treatment of vWD
- 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.
Bernard-Soulier syndrome
- 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.
Disorders of activation
- Storage pool deficiencies
- EX: gray platelets syndrome (α-granules def.)
Storage pool deficiency
- Storage pool deficiency can occur, w/ a deficiency of either dense granules or α-granules
Gray platelet syndrome
= Deficiency of α-granules is known
Disorders from lack of dense granules
- 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.
Disorders of aggregation
Afibrogenemia
Glanzmann thrombasthenia
Afibrogenemia
- 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
Defect in primary hemostasis in afibrogenemia
- due to lack of fibrinogen for binding to GPIIb-IIIa to allow platelet aggregation
Defect in secondary hemostasis in afibrogenemia
- due to lack of fibrinogen for formation of cross-linked fibrin
Glanzmann thrombasthenia
- 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
Drug effects in platelet disorders
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
Quantitative Platelet Disorders
Thrombocytopenia Immune thrombocytopenic purpura (ITP) Alloimmune thrombocytopenia Drug-induced immune thrombocytopenia Other non-immune-mediated causes of thrombocytopenia
Thrombocytopenia
- a low platelet count (less than 150,000/uL)
Mechanisms of Thrombocytopenia:
- decreased platelet production
- increased platelet destruction or consumption
- sequestration of platelets in the spleen
PLT count & effect
- 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
Thrombocytopenia due to decreased platelet production
- 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
Immune thrombocytopenic purpura (ITP)
- most common cause of thrombocytopenia
- formerly called idiopathic TP
- due to increased destruction
- two forms of ITP: acute and chronic
Immune thrombocytopenic purpura (ITP) – Mechanism of platelet destruction
- 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.
Acute ITP
- 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.
Chronic ITP
- 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
Treatment of Chronic ITP
- most commonly corticosteroids, IVIG, and splenectomy
- rituximab (anti-CD20) to deplete B cells
- TPO (thrombopoetin mimicker)
Effect of steroids in Chronic ITP
- work by dampening proliferation of B cell clone making the autoantibody
- effect seen in 7-10 days of starting treatment
Effect of IVIG in Chronic ITP
IVIG acts by blocking splenic Fc receptors to prevent their binding to antibody-coated platelets
- effect seen in 1-2 days
Effect of splenectomy in Chronic ITP
- Splenectomy works by removing site of autoantibody-induced platelet removal
- leads to lasting responses in 60-70% of patients
Alloimmune thrombocytopenia
- 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
Drug-induced immune thrombocytopenia
- 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
Other non-immune-mediated causes of thrombocytopenia include…
Include
- DIC
- Sepsis
- thrombotic thrombocytopenic purpura (TTP)
- hemolytic uremic syndrome (HUS)
Thrombocytopenia with these disorders is due to increased platelet consumption
Thrombotic thrombocytopenic purpura (TTP)
- 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
Large vWF multimers in TTP
- 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
Treatment of TTP
- w/ plasmapheresis to remove large vWF multimers & replace the missing ADAMTS13
Hemolytic uremic syndrome (HUS) vs. TTP
- similar presentation to TTP
- BUT tends to more often be associated with renal failure
- AND tends to occur more often in children
Hemolytic uremic syndrome (HUS) mechanism & treatment
- 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
Why to evaluate for bleeding disorder?
- Evaluate cause of acute or chronic bleeding
- Determine potential for excess bleeding prior to invasive diagnostic or surgical procedures
Patient evaluation for bleeding disorder should include…
- assessment of historical information
- physical examination
- performance of basic hemostatic screening tests
- further testing depending on results of initial assessment
Hx in Bleeding Disorder Evaluation
- 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?
Interpretation of Bleeding types – Brisk Bleeding from obvious trauma
- suggests a local vascular defect
Interpretation of Bleeding types –Prolonged / recurrent bleeding
- likely a more generalized hemostatic disorder
Interpretation of Bleeding types – Sudden resumption of bleeding from injured site
- raises possibility of excessive fibrinolysis or abnormal crosslinking of fibrin
Interpretation of Bleeding types – Multiple site bleeding
- suggests more severe, generalized hemostatic disorder
Interpretation of Bleeding types – Mucocutaneous bleeding
- 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)
Interpretation of Bleeding types – Soft tissue/joint/deep bleeding
- more consistent with a defect in secondary hemostasis (coagulopathy)
Interpretation of Bleeding types—Suspicious Nosebleeds
Suspicious of bleeding disorder if…
- Occur every 1-2 months
- Last longer than 10 minutes
- Involve both nares
- Require medical attention or transfusion
Findings on physical exam in bleeding disorder evaluation
- 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.
Age differences in Bleeding disorders
- 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
Basic screening tests when evaluating excessive bleeding can include:
- 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.
