vessel damage leads to these subsequent events

1. vasoconstriction to temporary cease bleeding; subendothelial collagen exposed

2. Platelets adhere to subendothelial collagen, a fibrous, negatively charged protein in connective tissue (adhesion)

3. collagen activates platelets to change into more of a 'star' shaped structure, and they stick to collagen

4. eventually, platelets begin sticking to each other (aggregation)

5. Platelets secrete granules that stimulate other platelets to begin coagulation (the mortar in a rock-wall dam)

6. eventually forms a hemostatic plug

Endothelial cells

-Provide a smooth, non-clotting surface so that blood flows freely in vessels

-Initiate clotting by exposure of subendothelial collagen

Blood Vessels

-prevent unwanted clotting -promote necessary clotting -contract directly after injury to prevent bleeding

-dilate later on to bring in necessary clotting factors

-assist with fibrinolysis when clot is no longer needed

1. maturation of committed stem cells (CFU-Meg) influenced by thrombopoietin.

2. nucleus of megakaryocytes matures before cytoplasm, resulting in multiploidy cells (up to 32N). Must be at 8N before they are morphologically recognizable megakaryocytes.

3. # of megakaryocytes is controlled by stimulation of CFU-GEMM by IL-3 and GM-CSF

Non-thrombogenic factors:

1. negatively charged surfaces repel platelets/hemostatic proteins

2. heparin sulfate inhibits thrombin via AT-III

3. Thrombomodulin inhibits thrombin coagulant activity

4. PGI2 (prostacyclin): vasodilation and inhibition of platelet aggregation

5. Tissue Plasminogen Activator activates fibrinolysis

6. Nitrous Oxide (NO): vasodilator

Thrombogenic

1. von Willebrand Factor stimulates adhesion

2. Tissue thromboplastin activates coagulation

3. Plasminogen Activator Inhibitor (PAI-1) inhibits fibrinolysis

4. Endothelin stimulates vasoconstriction

1. 4 to 16 times the number of chromosomes of any other cells. 2. increased granules in the cytoplasm causes a decreased basophilia and a pink hue.

3. Glycocalyx (extension of membrane proteins; absorbed proteins from plasma/receptors for mediators (VWF, Thrombin, firbinogen, epi, ADP)

4. Sol-Gel zone: has microtubules (made of tubulin) to maintain discoid shape (flying saucer) -tubulin=spectrin analogue -also contains actin/myosin-like proteins, like skeletal muscles

5. endomitosis (endoreduplication): nuclear division without cytoplasmic division

Describe the various anatomic features of the platelet, differentiating ones that are unique to the platelet vs. other typical cells.

Be able to link these structures/componenents to platelet adhesion/aggregation (structure/function relationships) and make predictions about what might happen as a result of alterations in these various structural components

* Organelle Zone
    <>Granules
         + Dense Bodies (non-protein platelet mediators)
             eg ADP, ATP, Ca2+
         + Alpha Granules (has a 'P': remember Proteins!)
             eg Proteins made by megakar's, as well as other cells; like VWF, factor V, plasminogen activator inhibitor-1 (PAI-1)

             # Plt-specif prot's eg. Platelet factor 4
             # Proteins absorbed from plasma, like fibrinogen, factor V, albumin

Differentiate among adhesion, aggregation, and agglutination as they would specifically relate to platelets.

Describe various circumstances that would impact one of these functions and not another and focus on the potential differences in the physiologic consequence.

Aggregation

describe

tell situation that would affect this

Aggregation can heal small inj alone

Platelets---Surfaces (in vivo: major surface is collagen)

CIRCUMSTANCE AFFECTING THIS: LOWERED CALCIUM

**Leads to reversal of shape change  caused by TxA2 (rel'd from activated platelets)Incomplete activation=PLT contributes nothing to clotting 

--Activation and release of granules stim's additional platelet involvement

--ADP in granules added to environment begins aggregation

AGGREGATION IS BIPHASIC

1. Primary: Loose due to weak agonists=Reversible

2. Secondary: Takes longer, requires stronger agonists (ADP, Collagen, Epi, TxA2); Irreversible

Agglutination

describe

what situation would affect this

Agglutination

• PLT--(Gp1b)--Ristocetin--(Gp1b)--PLT
• to assess the adhesion step of hemostasis, ristocetin is added, and binds to VWF--Gp1b complex, which cause adherable platelets to fall out of solution
  
• affected if no release of granules to initiate secondary aggregation
  

Describe the events that occur in adhesion and aggregation of platelets including stimuli of various events, differentiating platelet-related/produced mediators from non-platelet mediators.

1. Granule release thru canalicular system, which depends on ATP for energy and IC calcium.
2. Platelet Membrane Phospholipid (Platelet Factor 3): Essential to provide a surface for clotting to proceed
   

RR: 200-400 x 10³/µL

1. Low ( <50x10³/µL or 25x10³/µL)

=Thrombocytopenia->Clinical Spontaneous Bleeding (bruising, mucous membranes, heavy menses)

2. High (>1,000 x 10³/µL)

=Thrombocytosis->Bleeding

None of the platelets are stimulated by agonists to threshold amount

For the bleeding time test

a) describe the general procedure

b) interpret results and explain their significance, suggesting possible platelet functional defects. Describe the limitations of the test in terms of types of conclusions you can or can't draw

a. Modified Ivy Method

1. BP cuff pumped to 40 mmHg

2. Standardized incision 1mm in depth (length can vary)

3. Stop watch started as incision is made

4. Blood absorbed onto filter paper at 30s intervals (don't disturb clot)

5. Stop time when no more blood detected

b. Interpretation

When bleeding time is prolonged, it either suggests a decrease in platelet number or function

Limitations: cannot distinguish whether plt # is off or if function is off. Needs supplemental tests to diagnose. Only a screening test.

For platelet aggregation studies...

a. describe the general principle

b. recognize and differentiate among and draw normal curves for adp, epi, collagen and ristocetin

a. Principle:

Assesses whether agonist concentrations are correct in vivo by the presence of a biphasic curve

if conc's are too high will be a straight line

if too low will fall down after first increase

For platelet aggregation studies...(cont'd)

c. given a specific platelet defect, describe the changes in aggregation pattern you would expect in the case of each standard agonists (ADP, epi, collagen) 

d. interpret aggregation patterns across all three standard agonists providing a general analysis of the possible problem. understand the limitation of the interpretation.

c. Platelet Defects-- CHECK BOOK

1. ADP/Epinephrine: would show primary aggregation, but would slowly fall off after that because of platelets not secreting their granules

3. Collagen would stay flat low because of no activation of secondary aggregation (?)

d. Interpret aggregation patterns across all 3 standard agonists, providing analysis of the possible problem; understand the limitation of the interpretation

1. primary aggregation (1st increase) is due to the release of agonists, which activate platelets to release granules. If the platelet doesn't dump granules, the absorbances will fall back to where they were.

2. Secondary is caused by products released during the primary wave.

1. for Collagen and Ristocetin, only see single wave aggregation (ristocetin actually=agglutination, thru binding VWF attached to platelet surface Gp1b).

->Because aggreagation due to agonist is very strong, and the time between primary and secondary is so quick we don't see it.

1. Compare the count to RR
     thrombo-(up=-cytosis//dn=-penia)
2. Compare MPV to RR
    Increased//Normal
3. Morphology: describe any abnorm's
    -Size: large, enlarged, giant
        *correlate to MPV
    -Appearance
        *Granularity (eg Agranular)
    -Blebbing (yes/no)
    -"Bizarre" (not shape)

Aggregation

- Released TxA2 causes shape change, forming spheres with pseudopod projections

-Causes surface receptors to be exposed (GpIIb/IIIa=allows fibrinogen binding and formation of the clot backbone)

-Surface PPL's rearrange, allowing plasma coagulation

Platelet related/prod'd Mediators

-TxA2-->Shape change

-ADP in granules released to env. to begin aggregation

-Ca2+

Non-platelet mediators

-Collagen

-Epi

-ATP to help with granule release

-Ca2+

[image]

Describe the events that occur in adhesion and aggregation of platelets including stimuli of various events, differentiating platelet-related/produced mediators from non-platelet mediators.

14 cont'd

c. principle of PFA 100, interpret results

c. Principle of the PFA 100

-Automated in-vitro bleeding time instrument

-uses agonists (ADP, collagen, epi) on membranes in a capillary tube

-blood drawn thru capillary until it clots

-time it takes for a capillary to close=closure time

increases with poor platelet function