Pl a t el e ts

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PLATELETS

Maturation sequence of megakaryoblast takes about 5 days. Platelets are produced directly from the mekaryocytecytoplasm. As the megakaryocyte matures, clusters of granules aggregate to form platelets.

1. Megakaryoblast 20-50 um diameter

Blue cytoplasm

N/C ratio is about 10:1 Multiple nucleoli

Fine chromatin

2. Promegakaryocyte 20-60 um diameter

Less basophilic cytoplasm Chromatin becomes coarse

Irregularly shaped nucleus, may show showslight lobulation

N/C ratio is 4:1 to 7:1

3. Granular megakaryocyte

4. Mature megakaryocyte

40-120 um diameter Cytoplasm contains coarse clumps of granules

aggregating into little bundles, which bud offfrom the periphery to become platelets

Multiple nuclei are present No nucleoli is visible

N/C ratio is less than 1:1

5. Platelet/thrombocyte

1-4 um diameter Light blue to purple, very granular

o Chromomere – granular and located

centrallyo Hyalomere – surrounds the chromomere,

nongranular and clear to light blue

MORPHOLOGIC DIFFERENTIATION OF MEGAKARYOCYTIC CELL SERIES

Maturation Stage Cytoplasmic

Granules

Cytoplasmic

Tags

Nuclear Features Thromocytes

Visible

Megakaryoblast Absent Present Single nucleus, finechromatin, nucleoli

No

Promegakaryocyte Few Present Double nucleus No

Megakaryocyte Numerous Usually absent Two or more nuclei No

Metamegakaryocyte Aggregated Absent Four or more nuclei Yes

PLATELET STRUCTURE

Composed of 60% protein, 30% lipid, 8% carbohydrate, various minerals, water and nucleotides

Divided anatomically into four areas: peripheral zone, sol-gel zone, organelle and membranous system

1. Peripheral zoneGlycocalyxPlasma membrane

Submembranous area

2. Sol-gel zoneMicrofilaments: actin and myosinMicrotubules

3. Organelle zoneAlpha, dense granulesMitochondria, lysosomal granules

4. Membranous systemDense tubular systemOpen canalicular system

(surface connecting system)

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SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN

HEMOSTASIS

ROLE IN

HEMOSTASIS SUBSTANCE SOURCE COMMENTS ON PRINCIPAL

FUNCTION Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation

pathway Fibrinogen Alpha granules Converted to fibrin for clot formation

Factor V Alpha granules Cofactor in fibrin clot formation Factor VIII:vWF Alpha granules Assists platelet adhesion to subendothelium

to provide coagulation surface

Promote aggregation ADP Dense bodies Promote platelet aggregation Calcium Dense bodies Same Platelet factor 4 Alpha granules Same Thrombospondin Alpha granules Same

Promotevasoconstriction

Serotonin Dense bodies Promotes vasoconstriction at injury site Thromboaxane A2 precursors

Membrane phospholipids

Same

Promote vascular

repair Platelet-derived growth

factor Alpha granules Promotes smooth muscle growth for vessel

repair Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vessel

repair Other systems

affected Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis

2 – antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis

C1 esterase inhibitor Alpha granules Complement system inhibitor

HEMOST SIS

Process that retains the blood within the vascular system during periods of injury, localizes the reactions involvedto the site of injury, and repairs and re-establishes blood flow through the injured vessel

A system in dynamic balance that when tipped by deficiencies (congenital or acquired) of the procoagulant portionor excesses of the fibrinolytic portion, results in uncontrolled bleeding (hemorrhage); when tipped by deficiencies(congenital or acquired) of the fibrinolytic portion or uncontrolled activation of the procoagulant portion, the resultis excessive clot formation or persistence of clot (thrombosis)

SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS

ROLE IN

HEMOSTASIS SUBSTANCE SOURCE COMMENTS ON PRINCIPAL

FUNCTION Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation

pathway Fibrinogen Alpha granules Converted to fibrin for clot formation Factor V Alpha granules Cofactor in fibrin clot formation

Factor VIII:vWF Alpha granules Assists platelet adhesion to subendothelium to provide coagulation surface

Promoteaggregation

ADP Dense bodies Promote platelet aggregation Calcium Dense bodies Same Platelet factor 4 Alpha granules Same Thrombospondin Alpha granules Same

Promotevasoconstriction

Serotonin Dense bodies Promotes vasoconstriction at injury site Thromboaxane A2 precursors

Membrane phospholipids

Same

Promote vascularrepair

Platelet-derivedgrowth factor

Alpha granules Promotes smooth muscle growth for vesselrepair

Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vesselrepair

Other systemsaffected

Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis

2 – antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis

C1 esterase inhibitor Alpha granules Complement system inhibitor

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BASIC TERMINOLOGY FOR CLINICAL FINDINGS IN BLEEDING DISORDERS:1. Petechiae – purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary ability to withstand

normal blood pressure and trauma2. Purpura – hemorrhage of blood into small areas of skin, mucous membranes, and other tissues3. Ecchymosis – form of purpura in which blood escapes into large areas of skin and mucous membranes, but not

into deep tissues4. Epistaxis – nosebleed

5. Hemarthosis – leakage of blood into joint cavities6. Hematemesis – vomiting of blood7. Hematoma – swelling or tumor in the tissues or a body cavity that contains clotted blood8. Hematuria – rbc in urine9. Hemoglbinuria – hb in urine10. Melena – stool containing dark red or black blood

11. Menorrhagia – excessive menstrual bleeding

COAGULATION FACTORS1. Coagulation factors are also known as enzyme precursors or zymogens. They are found in plasma along with

nonenzymatic cofactors and calcium,2. Zymogens are substrates having no biologic activity until converted by enzymes to active forms called serine

proteases.

a. The zymogens include II, VII, IX, X, XI, XII and prekallikreinb. The serine proteases are IIa, VIIa, IXa, Xa, XIa, XIIa and kallikrein

3. Cofactors assist in the assist in the activation of zymogens and include V, VIII, tissue factor, and HMWK

4. In its active form, factor XIII is a transglutamase.5. Fibrinogen is the only substrate in the cascade that does not become an activated enzyme.

Blood factors are produced mostly in the LIVER and circulate in an inactive precursor form.

COAGULATION FACTOR NOMENCLATURE WITH PREFERRED NAMES AND SYNONYMS

NUMERAL PREFERRED NAME SYNONYMS

I Fibrinogen

II Prothrombin Prethrombin

III Tissue factor Tissue thromboplastinIV Calcium

V Proaccelerin Labile factor Accelerator globulin (aCg)

VII Proconvertin Stable factorSerum prothrombin conversion accelerator (SPCA)

VIII:C Antihemophilic factor (AHF) Antihemophilic factor globulin (AHG) Antihemophilic factor APlatelet cofactor 1

IX Plasma thromboplastin component (PTC) Christmas factor Antihemophilic factor BPlatelet cofactor 2

X Stuart-Prower factor Stuart factorPrower factor Autoprothrombin III

XI Plasma thromboplastin antecedent Antihemophilic factor C

XII Hageman factor Glass factorContact factor

XIII Fibrin stabilizing factor Laki-Lorand factorFibrinasePlasma transglutaminaseFibrinoligase

- Prekallikrein Fletcher factor

- High-molecular-weight kininogen Fitzgerald factorContact activation cofactor

Williams factorFlaujeac factor

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SCHEMATIC DIAGRAM OF

PHYSIOLOGIC HEMOSTASIS. Note thatthe ‘contact phase’ factors comprisingfactor XII, HK (high molecular weightkininogen) and PK (prekallikrein) are showngrayed; although factor XI can be activatedby this route under artificial in vitroconditions such as in the PTT test (see Fig.38-8 ), this pathway is not believed tocontribute to normal physiologichemostasis. Similarly, whereas Tissuefactor/VIIa can directly activate X to Xa inthe in vitro PT test under conditions whichsupra-physiological concentrations of

Tissue factor are employed, this reaction isshown as grayed because it does notcontribute significantly to clot formationunder normal in vivo physiologicalconditions. Normal clotting in vivoaccordingly is initiated when sufficientTissue factor/VIIa becomes available toactivate factor IX to IXa. Subsequently, IXain the presence of VIIIa activates X to Xa,which in turn activates prothrombin tothrombin in the presence of Va. Thrombinnot only then proceeds to clot fibrinogenand to activate platelets (see Fig. 38-2 ),

but additionally exerts critically important positive feedback by activating factors VIIIand V. Thrombin has further been showncapable of activating factor XI, thereby providing an additional pathway for theactivation of factor IX. PL (phosopholipid present on the surface membranes of platelets in vivo) and Ca

++ (calcium ions)

contribute to reactions as indicated.

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CHARACTERISTICS OF COAGULATION FACTORS

Factor Active Form PathwayParticipation

Vitamin KDependent

Present in BaSO4 Adsorbed Plasma

I Fibrin clot Common No Yes

II Serine protease Common Yes No

V Cofactor Common No Yes

VII Serine protease Extrinsic Yes No

VIII:C Cofactor Intrinsic No Yes

IXSerine protease

Intrinsic Yes No

X Serine protease Common Yes No

XI Serine protease Intrinsic No Yes

XII Serine protease Intrinsic No Yes

XIII Transglutaminase Common No Yes

Prekallikrein Serine protease Intrinsic No Yes

HMWK Serine protease Intrinsic No Yes

INHIBITORS OF COAGULATIONMajor site of inhibition: endothelium and platelet1. Protein C – degrades factor Va and VIIIa2. Protein S – degrades factor Va and VIIIa

3. Antithrombin III – major inhibitor of thrombin, also inhibits factors IXa, Xa, XIa, XIIa, kallikrein and plasmin4. Heparin cofactor II – inhibit thrombin

5. 2 macroglobulin – forms a complex with thrombin, kallikrein and plasmin, thus inhibiting their activities6. Extrinsic pathway inhibitor (EPI)

Lipoprotein assoc. coagulation inhibitor (LACI) – inhibits the VIIa-tissue factor complex7. C1 inhibitor – inactivator of factor XIIa and kallikrein, it also inhibits factor XIa and plasmin

8. 1 antitrypsin – inhibitor of thrombin, Xa and XIa

DISORDERS OF COAGULATION CAUSING CLOTTING FACTOR DEFICIENCIES

FACTOR INHERITED COAGULOPATHIES ACQUIRED COAGULOPATHY

INHERITANCE

PATTERN

COAGULOPATHY

I Autosomal recessive

Autosomal dominant

Afibrinogenemia

Dysfibrinogenemia

Severe liver disease

Diffuse intravascular coagulationFibrinolysis

II Autosomal recessive Prothrombin deficiency Liver diseaseVit K deficiency Anticoagulant therapy

V Autosomal recessive Factor V deficiency(OWREN’S dis, Labile factordef)

Severe liver diseaseDiffuse intravascular coagulationFibrinolysis

VII Autosomal recessive Factor VII deficiency Liver diseaseVit K deficiency Anticoagulant therapy

VIII X-linked recessive

Autosomal dominant

Hemophilia A

vWD

Diffuse intravascular coagulationFibrinolysis

IX X-linked recessive Hemophilia B Liver diseaseVit K deficiency Anticoagulant therapy

X Autosomal recessive Factor X deficiency Liver diseaseVit K deficiency Anticoagulant therapy

XI Autosomal recessive Hemophilia C(common in EasternEuropean Jewish descent/ Ashkenazi Jews)

*

XII Autosomal recessive Factor XII deficiency *

XIII Autosomal recessive Factor XIII deficiency Liver diseaseDiffuse intravascular coagulationFibrinolysis

Prekall Autosomal recessive Fletcher trait *

HMWK Autosomal recessive Fitzgerald trait *

* Unclear whether any acquired disorders cause factor XI or XII deficiencies or prekallikrein or HMK deficiency

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CLASSIFICATION OF VON WILLEBRAND DISEASE

TYPE DESCRIPTION

1 Partial quantitative deficiency of von Willebrand factor (vWF)

2 Qualitative deficiency of vWF

2A Decreased platelet-dependent vWF function with selective deficiency of high-molecular-weight multimers

2B Increased affinity for platelet glycoprotein Ib

2M Decreased platelet-dependent vWF function with high-molecular-weight multimers present

2N Markedly decreased binding of factor VIII to vWF

3 Complete deficiency of vWF

Lee and White clotting time methodEquipment: water bath 37

oC; glass test tubes 13 x 100 mm; stopwatch and plastic syringe (10 mL) and 20-

gauge needle. (Brown page 215)From Brown

Prothrombin Time:

Test tubes, 13 x 100 mm 0.1 mL patient’s plasma 0.2 mL (200 µL) thromboplastin-calciumreagent

APTT:

13 x 100 mm tube 0. 2mL plasma 0.2 mL APTT reagent 0.2 mLCaCl2 From Steiniger

Prothrombin Time:

0.1 mL plasma 0.2 mL (200 µL) PT reagent

APTT:

12 x 75 mm tube 0.1 mL PPP 0.1 mL APTT reagent 0.1 mL CaCl2

DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION SCREENING TESTS

Abnormal partial thromboplastin time (PTT) alone

Associated with bleeding: VIII, IX, XI defectsNot-associated with bleeding: XII, prekallikrein (PK), high-molecular-weight kininogen, lupus anticoagulants

Abnormal prothrombin time (PT) alone

Factor VII defects

Combined abnormal PTT and PT Medical conditions: anticoagulants, DIC, liver disease, vitamin K deficiency, massive transfusionRarely dysfibrinogenemias, factors X, V, and II defects

FAMILIES OF COAGULATION PROTEINS

THROMBIN-SENSITIVECOAGULATION PROTEINS

PROTHROMBIN FAMILY CONTACT FAMILY

I, V, VIII and XIIINot vitamin K dependentConsumed during coagulation

Absent in serum

Present in adsorbed plasma

II, VII, IX and XVitamin K dependentPresent in serum (except II)

Absent in adsorbed plasma

XII, XI, prekallikrein,HMWKNot vitamin K dependentPresent in serum

Present in adsorbed plasma

SUBSTITUTION STUDIES

DEF. PT APTT TT SUBSTITUTION STUDIES

Normal Plasma Adsorbed Plasma Aged Serum

I Abn Abn Abn C C NC

II Abn Abn N C NC NC

V Abn Abn N C C NC

VII Abn N N C NC C

VIII N Abn N C C NC

IX N Abn N C NC C

X Abn Abn N C NC C

XI or XII N Abn N C C CN – Normal NC – NOT CORRECTEDAbn – Abnormal C – CORRECTEDPT – Prothrombin TimeAPTT – Activated Partial Thromboplastin TimeTT – Thrombin Time

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CIRCULATING ANTICOAGULANTS

Prolonged APTT and PT not corrected Inactivate an activated coagulation factor or block interaction between coagulation factors and platelets Ex L upus inhib i tor

Nonsp anticoagulant IgG, IgM and IgA which interfere with phospholipid portion of the complex: Xa-Va-calcium-plt phospholipid

Platelet neutralization procedure Dilute Russell Viper Venom time

INSTRUMENTATION FOR TESTS OF HEMOSTASIS

1. Visual detection of fibrin clot formation

Tilt tube method

2. Electromechanical detection of fibrin clot formation

Fibrin strand formation is detected using a wire

loop or hook; has been incorporated into a semi-

automated mechanical instrument

Instrument: FIBROMETER

3. Photo-optical detection of fibrin clot formation

Detection of fibrin clot formation depends on the

increase in light scattering associated with the

conversion of soluble fibrinogen molecules to theinsoluble polymerized fibrin clot

Semi-automated instruments: Electra 750 and

750A, Fibrintimer series, and FP 910 Coagulation

Analyzer

Automated instruments: Ortho Koagulab 16S and

40A, the Coag-A-Mate X2 and XC, and the MLAElectra 700 and 800

FIBRINOLYSIS Digestion of fibrin clot, keeps the vascular system free of deposited fibrin/fibrin clot

Occurs when plasminogen is converted to plasmin

PLASMINOGEN ACTIVATORS1. Intrinsic activators

Factor XIIaKallikreinHWK

2. Tissue typeUrokinase-like PA

3. Therapeutic activatorsTreatment for thromboemboli

StreptokinaseUrokinaseTissue-like PA

INHIBITORS OF FIBRINOLYSIS

1. 2 antiplasmin ________________2. 2 macroglobulin3. Thrombospondin4. PA inhibitor 1 and 2

Degradation of cross-linked fibrin by plasmin Degradation of fibrinogen and non-crosslinked fibrinby plasmin

PATHOLOGIC FIBRINOLYSISPRIMARY FIBRINOLYSIS Excessive amounts of plasminogen activators from damaged cells/malignant cells Converts plasminogen to plasmin in the absence of fibrin formation

SECONDARY FIBRINOLYSIS DIC: uncontrolled, inappropriate formation of fibrin within the blood vessels

Infection; Neoplasm; Snake bite; HTR

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