1. Mrs. Ibtisam H. Alaswad Mr. Mohammed A. Jaber
Platelet Aggregation
Mrs. Ibtisam H. Alaswad
Mr. Mohammed A. Jaber

2. Lab Investigation of Primary Hemostasis
Platelets
Numbers
CBC
PLT count
PLT morphology
Function
Bleeding Time (BT)
Platelet Aggregation
Whole blood aggregation
Platelet-rich plasma aggregation
Islamic Unversity of Gaza
10/11/2010

3. Platelet Structure
Islamic Unversity of Gaza
10/11/2010

4. PLT Granules’ Content Granule Function Alpha Thromboglobulin(β-TG) PF4
PDGF
Fibrinogen, Factors V & VIII
vWF
Plasminogen
a1-antiplasmin
HMWK
Fibronectin
Inhibit heparin; vessel repair
Inhibit heparin
Vessel repair
Fibrin formation
PLT Adhesion
Precursor of plasmin (fibrinolysis)
Plasmin inhibitor
Contact activation: intrinsic coagulation path
Promotes PLT spreading
Dense
ADP/ATP
Calcium
Serotonin
PLT agonist
Regulates PLT activation
Promotes vasoconstriction
Lysosomes
Proteolytic, hydrolytic enzymes
Digest vessel wall matrix and debris
Primary hemostasis, Secondary hemostasis

5. Overview: Platelet Function
PRIMARY HEMOSTASIS
Form platelet plug: damaged endothelia
Nurture endothelia
SECONDARY HEMOSTASIS
Reaction surface for coagulation
Graphic accessed at URL

6. Platelet Plug Formation: Adhesion
Platelets bind to exposed adhesive subendothelial connective tissue
Collagen
vWF
Fibronectin
Mechanism components
vWF: links PLT to endothelial binding site
PLT receptor GPIb
Collagen fibers
Actin contracts & pseudopods form
REVERSIBLE
Facilitates activation

7. Platelet Activation After PLT adhesion A change in PLT shape
Generation of biologically active mediators
Degranulation
The specificity of PLT activation and signal transduction is maintained by the presence of PLT receptors that recognize the appropriate PLT agonists.
Thrombin
ADP
Islamic Unversity of Gaza
10/11/2010

8. Islamic Unversity of Gaza
10/11/2010

9. Platelet Plug Formation: Aggregation
Platelet-Platelet interaction
Mechanism components
ATP
Ionized calcium
Fibrinogen
PLT receptor GPIIb/IIIa
Initial aggregation – REVERSIBLE
Secondary aggregation – IRREVERSIBLE* = white clot, a.k.a platelet plug formed.
*The transformation of irreversible aggregated platelets into a mass of degenerative platelet material without membranes is termed viscous metamorphosis. (by platelet lysosomes)

10. Platelet Plug Formation: Secretion
Discharge of granules’ contents
Markers of PLT activation*
PF4
PDGF
Thromboglobulin (β-TG)
Promote & Amplify PLT activities
Primary hemostasis
Secondary hemostasis
*Markers of PLT activation these are proteins virtually absent from normal plasma and found in small concentrations within platelet α granules.
A number of clinical conditions such as arteriosclerosis, cerebrovascular disease, cardiopulmonary bypass, shock, venous thrombosis, and DIC are associated with increased plasma levels of these markers, thus signifying platelet activation.

11. Inherited Platelet Disorders- Qualitative
Qualitative disorders
Adhesion
Bernard-Soulier syndrome ( GP Ib-IX )
Platelet-type (Pseudo-) von Willebrand disease ( GP Ib receptor) *
Collagen receptor deficiency (GP VI)
Aggregation
Glanzmann thrombasthenia (Gp IIb-IIIa)
Secretion
Dense (δ) granule defects (storage pool deficiency)
α granule defects (gray platelet syndrome)
Platelet procoagulant activity
Scott syndrome PF3
*Platelet-type (also known as pseudo-vWD or platelet-type [pseudo] vWD) Platelet-type vWD is an autosomal dominant type of vWD caused by gain of function mutations of the vWF receptor on platelets; specifically, the alpha chain of the glycoprotein Ib receptor (GPIb). This protein is part of the larger complex (GPIb/V/IX) which forms the full vWF receptor on platelets. The ristocetin activity and loss of large vWF multimers is similar to type 2B, but genetic testing of vWF will reveal no mutations.
Islamic Unversity of Gaza
10/11/2010

12. Platelet Activation (signaling)
Islamic Unversity of Gaza
10/11/2010

13. Platelet Aggregometry
Platelet aggregation is an essential part of the investigation of any patient with a suspected platelet dysfunction.
Principle
We are using Aggregating agents to induce platelet aggregation or cause platelets to release endogenous ADP, or both.
Platelet aggregation is studied by means of a platelet aggregometer, Used Principle:
Photo-optical Method
Electrical Impedance Method
luminescence technology (Platelet Lumiaggregometry)
Islamic Unversity of Gaza
10/11/2010

14. Aggregating Agents (agonist)
Collagen*
ADP*
Epinephrine*
Arachidonic acid*
The antibiotic ristocetin*
Thrombin
Serotonin
Snake venoms, antigen-antibody complexes, soluble fibrin monomer complexes, and fibrin(ogen) degradation products (FDPs).
* The most using
Islamic Unversity of Gaza
10/11/2010

15. Electrical Impedance Method
These types of analyzers may use citrated whole blood, as the test sample.
As platelets aggregate, the coat an electrode, impeding the electrical current through the ana­lyzer.

16. luminescence technology (Platelet Lumiaggregometry)
The lumiaggregometer may be used to simultaneously measure platelet aggregation and secretion. The instrument records both aggregation and secretion of dense-granule ATP.
The ATP is measured by its reaction with firefly luciferin to give chemiluminescence. The resulting light emission is detected, amplified, and recorded by the instrument.
Performed by using whole blood or PRP.
This modification of aggregation is particularly sensitive to ATP release, and is as sensitive measure of platelet activation.

17. Photo-optical Aggregometry *PLT Aggregometry Using PRP
The Platelet-rich plasma, which is turbid in appearance, is placed in a cuvette, warmed to 37°C in the heating block of the instrument, and stirred via a small magnetic bar.
Baseline light transmittance through the platelet-rich plasma is recorded. The addition of an aggregating agent causes the for­mation of larger platelet aggregates with a corresponding increase in light transmittance, because of a clearing in the platelet- rich plasma. The change in light transmittance is con­ verted to electronic signals and recorded as a tracing by the chart recorder.

18. Photo-optical Aggregometry
Patient Sample – 3.2% citrated WB
Test Sample – PLT-rich Plasma
Principle – photometry: optical density of PRP warmed to 37° C is determined before and after the addition of various aggregating agents
Issues
Sample quality is critical
Fibrinogen levels are important
Agonists must be prepared fresh daily
Thrombocytopenia makes result interpretation difficult
Complete patient history is essential
Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is approximately 200 × 109/L, and platelets are maintained in suspension by a magnetic stir bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc., Havertown, PA.)
Islamic Unversity of Gaza
10/11/2010
Figure 2 – Five possible phases of PLT aggregation: 1) baseline, 2) agonist addition and shape change, 3) primary wave, 4) secretion, and 5) secondary wave.
Graphics accessed URL

19. Sample Platelet-Rich Plasma (PRP)
PRP is prepared and adjusted, if necessary, to a count of X 109/L by mixing with PPP.
Islamic Unversity of Gaza
10/11/2010
Graphics accessed URL &

20. Sample Quality for PRP PLT Aggregometry
Clean venipuncture
Hemolysis = increased plasma ADP = prematurely activated platelets
Lipemia may obscure OD during PLT aggregation
PRP contact with glass = prematurely activated PLTs
Keep samples capped to prevent loss of CO2 – change sample pH
Store room temp – prevent inhibition of PLT aggregation
Perform testing within 3 hours of sample collection
Islamic Unversity of Gaza
10/11/2010
Graphic accessed URL

21. PRP Aggregometry Agonist & Patterns
ADP (at appropriate concentration)
Biphasic curve: 1o and 2o waves (requires intact prostaglandin pathway)
Note: if ADP is added at too low a concentration or too high a concentration, will not get biphasic response
Epinephrine
Biphasic curve; requires intact prostaglandin pathway
Collagen
Lag phase followed by 2o wave only
Ristocetin
A biphasic however, often only a single broad wave
Binds to vWF/GPIb/IX complex and results in agglutination
Evaluates adhesion reaction
Ristocetin is an antibiotic, It causes platelet agglutination and blood coagulation and is used to assay those functions in vitro, e.g., to diagnose von Willebrand disease (vWD) or the Bernard-Soulier syndrome. Platelet agglutination caused by ristocetin can occur only in the presence of large multimers of von Willebrand factor, so if ristocetin is added to blood lacking the factor (or its receptor -- see below), it will not coagulate.
10/11/2010

22. PRP Aggregometry Agonist & Patterns
Thrombin
Biphasic curve. Irreversible aggregation only (does not require cyclooxygenase)
Arachidonic acid
2o wave only; assesses cyclooxygenase pathway
Serotonin
A primary wave of aggregation with a maximum of 10% to 30% transmittance followed by disaggregation.
Islamic Unversity of Gaza
10/11/2010

23. Interpretation
Platelet aggregation occurs as a two-step process, known as primary and secondary waves of aggregation.
The primary wave of aggregation is observed when platelets adhere to one another in the presence of an external agent (agonist) such as ADP, epinephrine, or ristocetin.
Secondary aggregation is characterized as the aggregation that occurs after the platelets have been stimulated to secrete the substances contained in their organelles.
It should be noted that some agonists will stimulate primary aggregation and some will stimulate secondary aggregation. Others will stimulate both primary and secondary aggregation, yielding a "biphasic" aggregation curve.
Islamic Unversity of Gaza
10/11/2010

24. Interpretation
In addition, different concentrations of the same agonist can produce varying patterns of primary and secondary aggregation.
For example,
low concentrations of ADP induce biphasic aggregation (i.e., both a primary and a secondary wave of aggregation);
very low concentrations of ADP (l.5 ug/ml. final concentration) induce a primary wave followed by disaggregation;
And high concentrations of ADP (10 ug/ml, final concentration) induce a single, broad wave of aggregation" (Fig.)
A biphasic aggregation response to ADP will not be seen in
patients with platelet release disorders.
Patients with Glanzrnann's thrombasthenia show incomplete aggregation with ADP regardless of the final concentration.
Islamic Unversity of Gaza
10/11/2010

25. Interpretation
In patients with severe von Willebrand disease, aggregation to ristocetin is characteristically absent. Decreased to normal aggregation to ristocetin can be seen in patients with mild von Willebrand disease. Correction of the abnormal ristocetin aggregation curves can be seen by the addition of normal, platelet-poor plasma to the patient's platelet-rich plasma.
Abnormal ristocetin-induced platelet aggregation may also occur in patients with
Bernard-Soulier syndrome,
Platelet storage pool defects
Idiopathic thrombocytopenia purpura (ITP).
Islamic Unversity of Gaza
10/11/2010

26. Glanzmann thrombasthenia
Normal PLT count, but abnormal clot retraction
Absence of secondary aggregation to ADP, epinephrine, collagen, (thrombin)
Normal response to ristocetin
Islamic Unversity of Gaza
10/11/2010

27. Bernard-Soulier syndrome
Platelet aggregation test
Failure to aggregate in the presence of ristocetin
Aggregation by other agonists (ADP, collagen, epinephrine): normal
Response to low-dose thrombin: may be delayed
Islamic Unversity of Gaza
10/11/2010

28. Platelet storage granule defects
Dense (δ) granule defects ~ storage pool deficiency
α granule defects ~ gray platelet syndrome
Heterogeneous group of disorders
Mild to moderate bleeding diathesis
Abnormalities in platelet aggregation
Islamic Unversity of Gaza
10/11/2010

29. Comment
In evaluating patients with suspected platelet disorders, the aggregating agents most commonly used are ADP in varying concentrations, collagen, epinephrine, and ristocetin, Aspirin, aspirin compounds, and anti-inflammatory drugs inhibit the secondary wave of aggregation by inhibiting the release reaction of the platelet.
Reduced or absent aggregation as well as disaggregation curves may be observed in patients taking medication containing aspirin. Other medications or substances have also been identified as inhibiting platelet function, such as ibuprofen, red wine, and a variety of herbs. Patients should be questioned carefully about possible ingestion of these substances before interpreting abnormal aggregation results.
Islamic Unversity of Gaza
10/11/2010

30. Comment
The intensity of platelet aggregation may be estimated by recording the change in absorbance as a percentage of the difference in absorbance between platelet-rich and platelet-poor plasma.
This has limited usefulness because absorbance is dependent on the size and density of platelet clumping and the number of platelets that aggregate.
Islamic Unversity of Gaza
10/11/2010

31. ADP agent
Islamic Unversity of Gaza
10/11/2010

32. Ristocetin & Serotonin Agent
Islamic Unversity of Gaza
10/11/2010

33. Collagen, Epinepherine & Thrombin Agents
Islamic Unversity of Gaza
10/11/2010

34. Platelet Response to Agonists
Addition of thrombin
(strong stimulation )
Addition of ADP
(mild stimulation)
Platelets - unstimulated
Characteristic discoid shape
Increased spreading, filaform process extension (arrows) and aggregate formation (stars)
Shape change (elongation and crescents) and filaform process formation (arrows)
Islamic Unversity of Gaza
10/11/2010

35. Platelet Aggregation to Thrombin
Islamic Unversity of Gaza
10/11/2010

36. Drugs and PLT Function Aspirin Clopidogrel Dipyridamole Abciximab
Acetylsalicyclic acid
Irreversibly inhibits Cyclooxygenase
Clopidogrel
Plavix
irreversibly inhibits P2Y12
Dipyridamole
inhibits Thromboxane synthase
Abciximab
ReoPro
inhibits GP IIb/IIIa
Brinkman WT, Terramani TT, Najibi S, Chaikof EL. Platelets: is aspirin sufficient or must we know how to pronounce abciximab? Semin Vasc Surg Dec;15(4):
(Pronounce: ab-SIKS-ih-mab)