Platelets & Hemostasis part 1 Flashcards
Define hemostasis.
What clotting factors are involved?
Stoppage of blood without obstruction of blood flow
–> Blood clotting factors include:
* Prothrombin
* Coagulation factors VII,
IX, X, XI, XII
What do we need for hemostasis to be successful?
We need a balance between coagulation and fibrinolysis. Want to stop blood from escaping vessels via coagulation factors.
As in the diagram shows, Hemostasis requires a balance between coagulation and
fibrinolysis. The multiple steps to reach the goal to stop the blood from scaping the
blood vessel is achieve by coagulation activation (generating thrombin that converts
fibrinogen into fibrin leading to the formation of the clot.
Who participates in this process? First there must be VASCULAR INJURY, then 1-
Vessel wall (vasoconstriction, activation of hemostasis), 2-platelets for the primary
hemostatic plug to form, and activation of coagulation pathway; 3-coagulation factors
are activated (fibrin plug formation, which is the SECONDARY hemostatic),
3
What makes hemostasis balanced?
The goal is to have a balance between enough anticoagulants and coagulants for
good hemostasis> not too much from one side or the other.
Imbalances in hemostasis can lead to?
Define primary hemostasis.
Primary hemostasis comprises: platelets and endothelium
While secondary hemostasis requires: clotting factors (coagulation cascade)
Define secondary hemostasis.
While secondary hemostasis requires: clotting factors (coagulation cascade)
What cells are pictured here?
Platelets
Label the image accordingly
Activated and non-activated platelets on a blood vessel
endothelia cell surface, scanning electron micrograph (SEM)
What can be seen below?
Platelets morphology
* 2-4um
* anucleated
* Blue cytoplasm & reddish cytoplasmic granules
* Alpha and dense granules
Label the image accordingly
What are Alpha granules?
Located within platelets
a) Alpha granules
* Seen in blood smears
* Contain coagulation factors & proteins:
* Fibrinogen,
* Factor V,
* vWF, thrombospondin,
* Platelet-derived-growth factor (PDFG)
What are Dense granules?
located within platelets
Thrombocytes (non-mammalian species)
* Equivalent to platelets in non-mammalian species:
* Round dense nucleus
* Clear cytoplasm (small amounts)
Megakaryocyte and platelet formation, scanning electron
micrograph (SEM).
Newly formed platelet after megakaryocyte differentiation,
scanning electron micrograph (SEM).
Platelets come from megakaryoblast, as these megakaryoblast “mature” to
megakaryocytes they become LARGE
Megakaryocytes are only present in Bone marrow
Where are platelets produced?
- Platelets are produced by megakaryocytes in bone marrow
What are Megakaryocytes?
§ Megakaryocytes are large cells with abundant cytoplasm
§ Present only in bone marrow sinus
§ they replicate the DNA without cell division (endomitosis)
What is an important physical characteristic of Megakaryocytes?
- Megakaryocyte have extensions called ”proplatelets” processes extend into sinus lumen –>
individual platelets - Maturation time: megakaryoblast to platelet release ~4-5 d
Thrombopoiesis is regulated by?
- Thrombopoiesis Regulated by: thrombopoietin (TPO)
- Production & differentiation
- Produced continually by: Liver, also kidney, muscle
- Platelets circulating lifespan 5-9 days
- 30-40% of circulating platelets mass is found in: spleen
Thrombopoietin (TPO) regulates the production and differentiation
TPO is produced by hepatocytes in liver, also comes from kidney and muscle
Platelets last in circulation about a week and interestingly 30-40% of circulating
platelets are found in the spleen
Activated platelets. Colored scanning electron micrograph
(SEM) of activated platelets attached to surgical gauz
What is the function of platelelts?
- Primary hemostasis (vasoconstriction and platelet plug formation)
a. Adhesion
b. Activation
c. Aggregation
d. Granule secretion
All to promote hemostasis - Secondary hemostasis
- Others: clot retraction, inflammation
There are 3 main functions: hemostasis (clot formation) and thrombosis, there are
other functions beyond hemostasis like Clot retraction, FYI: inflammation. We will
cover the secondary hemostasis on part 2 of hemostasis.
How are primary and secondary hemostasis related?
- These 2 processes happen simultaneously and are mechanistically
intertwined - Imbalances due to a defect –>:
- thrombosis (too much clotting)
- bleeding (not enough clotting)
If there is out balance due to a defect in on theses systems, then there will either be:
thrombosis (too much clotting) or bleeding (not enough clotting)
How long does it take for the primary hemostatic plug to form? Explain what happens.
Formation of this primary hemostatic plug takes ~3-5 min
* Platelets adhere to endothelium
* Platelets undergo activation
* Aggregate to form a platelet plug
So lets review this primary hemostasis process and what it involves from the platelet
to reach the goal of hemostasis
Simplified, primary hemostasis is the platelet adhesion and their activation to
produced a platelet plug to stop the bleeding.
How is this activated?
What are the requirements for Adhesion?
Adhesion
* Requires:
1. von Willebrand Factor (vWF)
- Binds to GPIb on Platelet surface
- Bridge between Platelets and collagen
2. Ca 2+
3. ADP
4. Thromboxane A2 (TXA2)
5. Thrombin
Blood vessel injury leads to platelets to be exposed to SUBendothelial matrix which is
what make the platelets adhere to the endothelial matrix, then once adhered, the
platelets are activated
This diagram attempting to represent the ADHESION.
The exposed to SUBendothelial is what makes the platelets to adhere to the
endothelial matrix.
This requires: 1-the GP1b that is present on the surface of the platelet to BIND with
the vWF
Abnormalities (such as congenital deficiencies) in various receptors or bridging
molecules lead to diseases indicated in the colored boxes.
What does the step “Activation” require?
- Shape changes
- Caused by exposure of collagen and release of Thrombin (Factor II )
- Secretion of granule products
- To activate more platelets
- This diagram attempting to represent the activation Now once platelets are
adhered and binding them to AGONISTS (especially collagen) activates
phospholipases to induce the synthesis of TXA2 (thromboxane A2 ). - The PLT activation requires a few things to happen to the actually PTL, such as:
shape changes (smooth to filopodia) and this shape change occurs under the
influence of Thrombin (factor II), this shape change helps to increase the surface
area - The secretion of granules occurs once the PTL are activated
TXA2 is important for the next step: aggregation as are also other products.
+++++++++++++++++++
Fibrinogen = Factor I
vWF = Factor VIII
27
What does platelet activation lead to?
Leads to:
* Recruitment of more platelets
* Further platelet activation
* Facilitated coagulation
* Mediate vessel repair
Remember that the main goal of the hemostasis is to stop the bleeding!
What does the step “Aggregation” require?
CATT
Requires:
* ADP
* TX-A 2 (Irreversible platelet aggregation)
* Thrombin
- Product of Secondary hemostasis
- Positive feedback
* Ca2+
The thromboxane A2, induces an irreversible PTL aggregation
Thrombin = Coag. Factor II
What functions do platelets serve beyond homeostasis?
- Clot retraction
* Contract via actinomyosin filaments
* Facilitates:
- Wound closure Vessel patency
FYI
1. Inflammation
* Chemotaxis, enhances neutrophil function Antimicrobial activity
* Source of:
- Inflammatory mediators, Vasoactive substances, Mitogenic agents
Can you recall the order of the platelet plug
formation
* A. Aggregation → Activation → Adhesion
* B. Activation → Aggregation → Adhesion
* C. Adhesion → Activation → Aggregation
* D. Aggregation → Adhesion → Activation
The answer is? C
Platelets adhere to endothelium
Platelets undergo activation
Aggregate to form a platelet plug
What lab tests allow us to evaluate a patient’s platelets?
- PLT concentration and morphology (size)
* Blood smear
* Hematology analyzers - Production
* Bone marrow aspirate (BMA) - Function
* Bleeding time tests
* Specific platelet function tests
How do we evaluate platelet concentration?
- Two methods for evaluation
1. Evaluation on a blood smear
2. Hematology analyzer - Measured and typically part of the routine CBC
Collect blood into EDTA tubes (purple top)
Venipuncture technique is important
Why is important to use anticoagulant?
TO avoid activation of the platelets and of course we are trying to count how many
are in circulation!
How can we count the number of platelets in a blood smear?
- Count number of platelets in ten (10) HPF (100X)
- Calculate the average
- Multiply the average by 20,000 à approx. platelet concentration/uL
Check for PLT clumps, especially in cats and cattle
Clumping of platelets could alter the number of platelet counts. And the report generated will say DECREASED and this will be a false thrombocytopenia (decreased
PLT)
- Platelets are smaller than RBCs
- But there is size variation
- More in cats
- Pale-staining in horses
- Macroplatelets > size of a RBC
Increased numbers of enlarged PLTs suggests active
production of platelets
Platelet regeneration
How many platelets are in this HPF field?
- How many platelets are in this HPF field?
answer: n=5 - Number of platelets in a total of 10 fields:
- Average = 9+5+7+6+5+8+7+7+6+5
10 - Approx. platelet concentration = 6.5 * 20,000 =
130,000/uL
=6.5
What does a hematology analyzer do?
- Counts thousands of PLT
- Categorized by their small size
- Therefore, platelets counts may be altered if
- Large numbers of macroplatelets
- Platelet clumps
What is the mean platelet volume?
- Mean Platelet Volume (MPV)
- Platelets are sized by the analyzer; counts many platelets and
calculates the average size - Increased MPV suggests increased thrombopoiesis.
- Platelets are sized by the analyzer; counts many platelets and
What are platelet production tests used for?
- Used in cases of thrombocytopenia
- Evaluated by Bone marrow aspirate (BMA)
- Megakaryocyte number and morphology
How would you interpret Thrombocytopenia ?
- Lower PLT count than lower RI
- Mild 80,000/μL-LRI (lower R.I.)
- Moderate 30,000-80,000/μL
- Severe <30,000/μL
Patients are at risk for
spontaneous hemorrhage
when the platelet count is <30,000 /μL
How would you interpret Thrombocytosis?
- Higher ____ count than upper __
PLT, RI
What are the clinical signs of thrombocytopenia?
- Platelet-associated bleeding pattern
- Bleeding from mucosal membranes
- Epistaxis
- Gingival bleeding
- GI bleeding
- Hematuria
- Petechia or ecchymosis
- Prolonged bleeding post a wound/Sx
- Excessive cutaneous bleeding
Epistaxis = nose bleeding
Hematuria = RBCs in urine
Petechia - thrombocytopenia
Thrombocytopenia
Not a specific disease
* Spontaneous hemorrhage
ØPLT count <20,000/uL
What are the causes of Thrombocytopenia?
- Decreased production by the bone marrow
- Increased destruction
- Sequestration
- Increased consumption/use
- Pseudothrombocytopenia
What conditions –> decreased production of thrombocytes –> thrombocytopenia ?
- Pure megakaryocytic hypoplasia (dogs)
- Bone marrow panhypoplasia
- Typically in conjunction with leukopenia (before
thrombocytopenia) - Drugs: estrogen, chemotherapy
- Toxins (mycotoxins)
- Radiation
- Typically in conjunction with leukopenia (before
- Myelophthisis is defined as?
- Myelophthisis causes Thrombocytopenia via?
- What are the different forms of Myelopthisis?
- What infectious agents can also lead to Thrombocytopenia?
- Myelophthisis is a form of bone marrow failure due to replacement of hematopoietic tissue by abnormal tissue.
- Decreased production
- Disseminated inflammation,
* Primary Neoplasia (leukemia)
* Secondary neoplasia: mast cell tumors, histiocytic sarcoma
* Myelofibrosis or myelonecrosis
- Disseminated inflammation,
- Infectious agents:
* FeLV/FIV,
* EIA,
* BVD
How does Immune-mediated thrombocytopenia leads to Increased destruction of thrombocytes?
Immune-mediated thrombocytopenia
a) Primary
* Antibodies against platelet antigens, e.g. GPIIb/IIIa
b) Secondary
* Antibodies secondary to another underlying condition
§ SLE
§ Neoplasia
§ Drugs
§ Vaccines – distemper, parvovirus, panleukopenia
Systemic lupus erythematosus (SLE): autoimmune disease where the immune system
attacks its own tissues: causing spread inflamm. And tissue damage in different
organs
What are the expected test results for Thrombocytopenia?
Expected Tests results:
* CBC:
* severe thrombocytopenia,
* +/- acute hemorrhagic anemia (regenerative, hypoproteinemia)
* +/- leukocytosis
* Coagulation Tests (PT, PTT): Normal
* Bone marrow aspirate: Increased megakaryocytes
* Serum Biochemistry: normal
What are the clinical signs of Immune-mediated thrombocytopenia?
Clinical signs
* Platelet-associated bleeding pattern
How does Sequestration lead to thrombocytopenia?
- Sequestration in large vascular beds
* Splenomegaly, splenic torsion, neoplasia
* Hepatomegaly, portal hypertension
* Vasodilation in endotoxic shock
* Severe hypothermia - The thrombocytopenia is mild to moderate
How does Increased PLT consumption cause Thrombocytopenia?
- Common causes:
- DIC
- Vasculitis (Ricketssial disease, FIP)
- Degree of thrombocytopenia: Mild to moderate, Rarely severe
- Hemorrhage occurs if there are coagulation defects (DIC) or leakage
of blood from vessels (vasculitis)
Pseudothrombocytopenia
- Analyzer does not measure platelets because
- Too big: macrothrombocytes
- Clumped
- Macrothrombocytopenia is a condition common in which species?
- This condition is caused by a mutation in?
- What are the clinical signs?
Cavalier King Charles Spaniel dogs
* Mutation in B1-tubulin gene – alters megakaryocyte proplatelet
formation and release
* No clinical signs or bleeding tendencies
* Total platelet mass (plateletcrit) is normal compared to a healthy
dog
* Platelets are functionally normal
* Giant platelets (high MPV) with low platelet coun
The Platelet Function test tests what?
What does it NOT test?
Bleeding test
* Test the ability of platelets to form a platelet plug
* Does NOT test fibrin plug formation
* Buccal mucosal bleeding time (BMBT)
* Abnormal (prolonged) when
* 1-decreased platelet function
And/or
* 2-decreased platelet numbers
Buccal mucosal bleeding time (BMBT)
* Make a standardize incision, blot blood gently, and measure time for
bleeding to cease.
* Time to clot formation varies by species and the size of the lancet.
Decreased platelet numbers or decreased platelet function will Increased the BMBT
vWD (von Willebrand disease) which means have abnormal vWF structure as well as
amount. Also in patients with thrombopathia like the inherited in Chediak Higashi
syndrome in cats or acquired disease
What is considered normal buccal mucosal bleeding time (BMBT) in:
cats
dogs
horses and cattle
In general, normal is considered:
* 1-3 min in cats
* 2-4 min in dogs
* 8-10 min in horses and cattle
>4 min indicative of vWD def or other thrombocytopathy
When to use BMBT?
- Patients with normal platelet count with concurrent clinical signs of
primary hemostasis disorder- Must rule out first possible coagulopathy (secondary hemostasis)
- Not needed in thrombocytopenic patients
- does not add information
Mechanisms for thrombocytosis
- Increased production
- Increased distribution in plasma
As mentioned already, thrombocytosis is the increased concentration of platelets
Thrombocytosis due to
* Physiologic
- Epinephrine-induced splenic contraction
- Excitement/fright
- Mild to transient thrombocytosis
Thrombocytosis due to
* Reactive (inflammatory)
- Results from inflammation: IL-6 (pro-inflamm. Cytokine)
- Stimulates TPO release –> increased platelet production
Thrombocytosis due to
* Recovery from previous thrombocytopenia
- Rebound response
- Increased bone marrow megakaryocyte production
Thrombocytosis due to
* Iron def.
- Uncommon
- Inconsistent finding
Thrombocytosis due to…
* Post-splenectomy
- Increased thrombopoiesis
- Increased TPO production
Thrombocytosis due to
* Essential thrombocythemia & Megakaryocytic leukemia
- Extremely rare
- Must evaluated bone marrow to Dx
- Rule out other causes first
Diseases of primary hemostasis
* Platelet function disorders (thrombocytopathies)
* Often ______ platelet concentration on CBC, abnormal ______
a. von Willebrand disease (vWD)
* Most common Inherited disorder in ___
* Name the dog breeds predisposed?
- Could be acquired:
- Excessive high ___ stress conditions: aortic ____, ______ dzs
normal, function
dogs, Doberman pinscher, German shorthair pointer, Chesapeake Bay Retriever, Scottish Terrier
shear, stenosis, cardiovascular
Clinical features of vWD
* ____ to _______ bleeding
- Exacerbated by _____ or ______
- Signs decrease with ___ and successive _____
–> Platelet/coagulation evaluation:
* Platelet count: _____
* BMBT: _______
* IF Suspected vWD, analyze _____ for wWF concentration
* PTT/ACT usually _____ (but can be prolonged if Factor ____ def. is pronounced
Clinical features of vWD
* Mild to severe bleeding
- Exacerbated by surgery or trauma
- Signs decrease with age and successive pregnancies
–> Platelet/coagulation evaluation:
* Platelet count: normal
* BMBT: prolonged
* IF Suspected vWD, analyze plasm for wWF concentration
* PTT/ACT usually normal (but can be prolonged if Factor VIII def. if
pronounced
What tests would you run to check for vWD?
- ELISA (most common)
- Immunoelectrophoresis: Determines relatives amounts of different sized vWF multimers
- Collagen binding activity assay: ELISA –detects only vWF that can bind to collagen
Diseases of primary hemostasis
b. Glanzmann thrombasthenia
* Def. of ?
* Platelets unable to bind _______ –>
no PLT ________ and clot ______
* Common in which dog and horse breeds?
GPIIb-IIIa, fibrinogen, aggregation, formation
Otterhounds, Great Pyrenees, quarter
horse, Thoroughbred, Peruvian paso
What is the clot retraction test?
- Measures the amount of time it takes for a clot to retract from a test tube
after thrombin is added - Clot retraction graded from 1+ to 4+
- Indicated in animals with a normal platelet count, but questionable
platelet function (similar to BMBT test) - Abnormal retraction seen in dogs and horses with Glanzmann
thrombasthenia
