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

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

Platelet Structure Islamic Unversity of Gaza 10/11/2010

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

Overview: Platelet Function PRIMARY HEMOSTASIS Form platelet plug: damaged endothelia Nurture endothelia SECONDARY HEMOSTASIS Reaction surface for coagulation Graphic accessed at URL http://www.medicine.mcgill.ca/physio/209A/Blood/blood6a.htm, 2007.

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

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

Islamic Unversity of Gaza 10/11/2010

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)

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.

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

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

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

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

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.

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.

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.

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 http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.

Sample Platelet-Rich Plasma (PRP) PRP is prepared and adjusted, if necessary, to a count of 200-300 X 109/L by mixing with PPP. Islamic Unversity of Gaza 10/11/2010 Graphics accessed URL http://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpg, http://www.accumetrics.com/images/img_product_overview.jpg, & http://cmed-tech.com/graphics/platelet2.jpg, 2009.

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 samples @ 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 http://labmed.ucsf.edu/labmanual/mftlng-mtzn/img/BD_plastic_citrate_draw.gif, 2009.

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

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

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

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

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

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

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

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

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

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

ADP agent Islamic Unversity of Gaza 10/11/2010

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

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

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

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

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. 2002 Dec;15(4):245-55. (Pronounce: ab-SIKS-ih-mab)