CP Blood Coagulation

8/4/2019 CP Blood Coagulation

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BLOOD COAGULATION

Functions: Maintain blood in a fluid, clot-free state in normal vessels Rapid and localized hemostatic plug at the site of injury

Abnormalities of clot formation may take two main forms: Failure to clot normally Failure to prevent excessive clotting

Regulated by three general components:1.Vascular wall2. Platelets3. Coagulation cascade

Diagrammatic presentation of normal homeostatic process

A.VASOCONSTRICTION:Shows that there is a site of injury,and in a period of arteriolar vasoconstriction occurs mostlyas a result of neurogenic mechanisms and is improved bythe secretion of local factors such as endothelin (a potent

endothelium-derived vasoconstrictor).

B.PRIMARY HEMOSTASIS:Shows that there is adhesion of platelets at the site of endothelial injury, and dramaticchange in shape (from small rounded disks to flat plateswith markedly increased surface area) and the release of secretory granules such as adenosine diphosphate (ADP)and thromboxane A2 (TXA2). Next, there will berecruitment of additional platelets (aggregation) to form ahemostatic plug primary hemostasis.

C. SECONDARY HEMOSTASIS:At the endothelial injury,tissue factors are also exposed; such that are factor III andthromboplastin , a membrane-bound procoagulantglycoprotein synthesized by endothelium. It acts inconjunction with factor VII as the major in vivo pathway toactivate the coagulation cascade leading to culminatingthrombin generation. Remember: thrombin cleavescirculating fibrinogen into insoluble fibrin , creating a fibrinmeshwork deposition; also induces further platelet recruitment and granule release. As a result, the formationof fibrin polymerization as the secondary hemostasissequence.

D. ANTITHROMBOTIC COUNTER REGULATION:Here itshows the fibrin polymerization and platelet aggregationform a solid permanent plug to prevent for furtheradditional damage or hemorrhage. Moreover, there is arelease of counter-regulatory mechanisms, such as of tissue plasminogen factor (t-PA, a fibrinolytic product) andthrombomodulin (blocks coagulation cascade) to limit theformation of further increase of the hemostatic plug to theinjury site.

Subject: PathologyTopic: CP-Blood CoagulationLecturer: Dr.PascualTranscriptionist:Editor:Pages: 12

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ENDOTHELIUM

Endothelial cells have the ability to restrain many aspects of normal hemostasis (stopping of blood flow).It can facilitate as antithrombotic and prothrombotic, whether there can be a thrombus formation, propagationor dissolution occurs. It has antiplatelet effect, anticoagulant and fibrinolytic effects wherein they inhibit thecoagulation cascade discussed above, on the other hand it also has a procoagulant entity, in which the plateletfactors (vWF), and antifibrinolytic affects are activated therefore, there will be an inhibition of t-PA (which can

limit the increase of hemostatic plug at the injury site).

Antithrombotic properties

Antiplatelet effectsDuring coagulation, platelets are activated and endothelial cells are stimulated by several

factors such as thrombin and cytokines to produce endothelial prostacyclin (PGI2) and Nitric Oxide (NO), both of these are vasodilators and inhibitors of platelet aggregation thus, there will be no plateletadhesion to the surrounding uninjured endothelium and so the inactivated platelets. Endothelial cellsalso elaborate ADPase , further inhibits platelet aggregation.

Anticoagulant effectsThey are mediated by membrane-associated, heparin-like molecules and thrombomodulin. Both

of them act indirectly, the former are cofactors that allow antithrombin III to inactivate thrombin,factor X and several other coagulation factors. The letter binds with thrombin; converting it from aprocoagulant to an anticoagulant capable of activating the anticoagulant protein C, this in turn inhibitsclotting through proteolytic cleavage of factors Va and VIIIa; and requires protein S, synthesized byendothelial cells, as a cofactor.

Fibrinolytic effectst-PA is synthesized by endothelial cells and promotes fibrinolytic activity to clear fibrin deposits

from the endolethelial surfaces.

Prothrombotic properties

Platelet effectsWhenever injury is encountered, it always result to platelet adhesion, may it be through vWF ,

which is an essential cofactor for binding platelets to collagen and other surfaces for both circulatingand collagen bound, is synthesized largely by endothelium. If there is a loss of the endothelium itexposes the deposited vWF and allows the circulating vWF to also bind to the basement membrane

Procoagulant effectsCytokine s [e.g. tumor necrosis factor (TNF) or interleukin-1 (IL-1)] and bacterial endotoxin all

induces endothelial cell production of tissue factor . It activates the extrinsic clotting pathway.

Anti-fibrinolytic effectsAgain, endothelial cells produce plasminogen activator inhibitors (PAIs) that depress

fibrinolysis.

Here are some factors that affect the endothelium per se:

Antithrombotic properties: Antiplatelet effects Anticoagulant effects Fibrinolytic effects

Prothrombotic properties: Platelet effects

Procoagulant effects

Anti-fibrinolytic effects


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PLATELETS Ultrastructure

Glycocalyx: Outer membrane surface rich in glycoproteinso Glycoprotein Ib and Factor IX: receptor for vWFo Glycoprotein IIb and IIIa: fibrinogen

Exposed by: Stimulation of thrombin ADP

o Glycoprotein Va: receptor for thrombin Kinetics

Maturation Megakaryoblast (progenitor cells) Mature megakaryocyte Mature Megakaryocyte

o Platelet fields: clusters of pink granules in the cytoplasmo Individual platelets are shed from cytoplasm into marrow sinuses then released into the

vascular lumeno mature megakaryocyte = 2000 7000 plateletso Normal circulation life: 7 10 dayso Removed by macrophage in the liver and spleen or by active use in coagulation mechanisms

Clinical findings of Patients with Platelet Abnormalities typically presents with: Skin and mucosal bleeding patterns

Petechiae Ecchymoses Epistaxis Menorrhagia

Patients who present with immediate postsurgical bleeding such as:Hemarthrosis, deep muscle, joint, intracerebral or retriperitoneal bleeding, anddelayed postsurgical bleeding (1 4 hours after) suggest a defect in theCOAGULATION CASCADErather than a PLATELET ABNORMALITY.

Abnormalities of Platelets can either beQUANTITATIVE or QUALITATIVE

Quantitative - abnormal platelet count Qualitative - abnormal platelet function

- can be surface membrane defect or a granule defect

QUANTITAVE

THROMBOCYTOPENIA decrease in the number or circulating platelets

Defective production in the bone marrow1. Decrease number of megakaryocytes due to :

congenital disorders acquired disorders (radiation, alcohol, thiazide diuretics, chloramphenicol, cancer

chemotherapy) marrow replacement by malignant cells (* MYELOPHTHISIC ANEMIA)

2. Ineffective platelet production due to : hereditary thrombocytopenia Megaloblastic anemia problem in B12/folate deficiency -> derrangement of DNA

synthesis -> ineffective production of platelets

Erytholeukemia (Di Gugliemos syndrome) - an acute myelocytic leukemia FABclassification, M6

Paroxysmal Nocturnal Hemoglobinuria (PNH)

.

Platelets Normal/Range Values:150-450 x 10 9/L150,000 450,000/mm 3

5-20 /OIF on peripheral smear

From the book: PNH blood cells are deficient in three GPI-linked proteins that regulate complementactivity:(1) CD55- decay accelerating factor(2)CD59 - membrane inhibitor of reactive lysis; a potent inhibitor of C3 convertase that prevents

the spontaneous activation of the alternative complement pathway-most important factor

(3) C8 binding protein


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A. Disorders of the Distribution and Dilution of platelets in the circulation Splenic pooling: splenomegaly, hypersplenism * platelets are removed by macrophages in the

spleen Hypothermia: vascular shunting Dilution in the circulation: transfused stored blood

B. Disorders that result in Destruction of platelets1. Combined consumption of both platelet and coagulation factors

e.g. DIC and its causes

2. Isolated consumption of platelets

THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP) caused by vascular wall dysfunction -> excessive deposition of platelet aggregates in

renal and cerebral vessels Vascular endothelial cell damage, increase in PAF, deficiency of PAF-inhibitor, decrease

PGI2, absence of t-PA FEMALE more affected than males Px will present w/ : microangiopathic hemolytic anemia, thrombocytopenia, neurologic

symptoms, fever, renal disease

HEMOLYTIC UREMIC SYNDROME

Symptoms similar to TTP but less pronounced neurologic symptoms and morepronounced renal symptoms

FRAT fever, renal disease, anemia, thrombocytopenia

VASCULITIS

HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count) Associated with pregnancy or delivery common in pre-eclamptic or eclamptic patients Microangiopathic hemolysis

3. Immune destruction of platelets

a.) IDIOPATHIC (IMMUNOLOGIC) THROMBOCYTOPENIC PURPURA (ITP) -type 2 hypersensitivity reaction

Acute ITP children 2-6 years of age sudden onset of thrombocytopenia following viral infections (rubella, chickenpox, CMV,and toxoplasmosis

Lasts for 2-6 weeks with a spontaneous remission in 80% of patients Platelet count plateletautoantibodies (most often IgG)

Chronic ITP adults 20-40 years old Female > male Insidious onset: can last for months to years Platelet count: 30,000 80,000/mm3 Tx: splenectomy (to decrease the number of macrophages in the spleen)

*mnumonic ni doc: FAT RN (fat nurse)Fever, Anemia Thrombocytopenia- Renal, Neurologic

From the book : DISSEMINATED INTRAVASCULAR COAGULATION (DIC)- an acute, subacute, orchronic thrombohemorrhagic disorder characterized by the excessive activation of coagulation,which leads to the formation of thrombi in the microvasculature of the body .


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b) POST TRANSFUSION PURPURA- antiplatelet antibodies produced by recipient of platelet

transfusions

c) DRUG-INDUCED ANTIBODY FORMATION :Quinidine and Heparin(mechanism : drug attaches to platelet membrane surface -> alter platelet membrane

protein -> develop Abs against platelets -> thrombocytopenia corrected after withdrawal of drug )

II. THROMBOCYTOSIS Bleeding or thrombotic complications Increase in circulating platelet count

2 types:Essential Thrombocytosis commonly occurs with inc.levels of other hematopoietic cells

Result of primary bone marrow disorder Often caused by a clonal proliferation that affects all hematopoietic cells Increased bleeding tendencies because of possible accompanying functional abnormalities Seen in Hodgkins disease, polycythemia vera (very high RBC count), myelofibrosis, chronic

myelogenous leukemia, AML- M7 type ( acute megakaryocytic)

Secondary Thrombocytosis- occurs in assoc. with massive blood loss Iron deficiency anemia assoc. with chronic blood loss Chronic inflammatory disease Rebound thrombocytosis occurs after platelet depletion through a massive blood loss Post-splenectomy no sequestration of platelets so values will increase

QUALITATIVE

1. Surface membrane defectsBERNARD SOULIER DISEASE

Functions disorder which may appear similar to ITP Decrease in platelet vWF receptor (glycoproteins 1b and IX) Bernard-soulier Vs Von Willebrand disease

GLANZMANNS THROMBASTHENIA Platelets are normal in number and appearance

Decrease in surface glycoproteins IIb and IIIa and a reduction in numbers of available fibrinogen binding sites

End result: disorder of platelet-platelet interaction* platelets are able to recruit other addtional platelets to the hemostatic plug but it cannot form a permanent plug because you it doesnt have binding sites for FIBRINOGEN

2. Abnormalities in granular fraction of platelets

DENSE GRANULES

Hermansky-Pudlak Syndrome- decreased number of platelet dense granules

Chediak Higashi Syndrome: - autosomal dominant- presence of giant lysosomes in the cytoplasm of ALL precursor bloodcells --> dense granule destruction

Wiskott-Aldrich Syndrome: sex linked, decreased in the number of dense granulescharacterized by : eczema ,thrombocytopenia and immunodeficiency

ALPHA GRANULES (Gray Platelet Sydrome )-aggregation and release properties diminished

Bernard-Soulier Disease Von Willebrand Disease

vW FACTOR normal decreasevW RECEPTOR

(glycoproteins 1b and 9)decrease normal


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3. Deficiencies of thromboxane generation: deficiency of cyclo-oxygenase enzyme4. Acquired

QUANTITATIVE QUALITATIVETHROMBOCYTOPENIA

Di Guglielmos Syndrome Thrombotic Thrombocytopenic Purpura

Hemolytic Uremic Syndrome Vasculitis HELLP Idiopathic (Immunologic) Thrombocytopenic Purpura

Surface membrane defetcs1. Bernard-Soulier Disease2. Glanzmanns Thrombasthenia

THROMBOCYTOSISEssential Thrombocytosis

Hodgkins Disease Polycythemia Vera Myelofibrosis Chronic myelogenous Leukemia AML M7

Secondary Thrombocytosis Iron Deficiency Anemia Chronic Inflammatory Disease Rebound Thrombocytosis Splenectomy

Abnormalities in granular fractions of plateletsDense Granules:

1. Hermansky-Pudlak Syndrome2. Chediak-Higashi Syndrome3. Wiskott-Aldrich Syndrome

Alpha Granules: Gray Platelet Syndrome

Laboratory Evaluation of Platelets *check for quantitative abnormalities FIRST

1. Platelet count Normal Values : 150,000 400,000/mm3

5-20 platelets/ oil immersion field on peripheral smear

2. Bleeding time Indicator of platelet abnormality in number or function Abnormal if platelet count is less than 100,000/mm3 and prolonged in cases of

Glanzmanns thrombasthenia, uremia and myeloproliferative syndromes, aspirin intake,vonWillebrand disease

PROCEDURE:Use of spring lancet producing a 5mm long and 1.0mm deep cut Normal values: 2-8 minutes

3.Torniquet test Demonstrate capillary abnormality due to intrinsic defect in the capillary walls (capillary

fragility) or to some type of thrombocytopenia

4.Clot retraction Begins 1 hr. and completes by 24 hrs. (a firm clot with 50% serum and 50% clot) Deficient clot retraction in all types of thrombocytopenia and in Glanzmanns thrombasthenia

COAGULATION CASCADE

Intrinsic Pathway Triggered by damage to the endothelial lining of blood vessel When blood comes in contact with certain foreign bodies Evaluated by activated Partial Thromboplastin Time (aPTT)

Extrinsic Pathway Triggered by tissue thromboplastin (Factor III) Released by damage endothelial and body tissues Evaluated by Prothromin Time (PT)

Mnemonics:Intrinsic = pItt

Extrinsic = pEt


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The figure shows the intrinsic &extrinsic pathways and the final common pathway.

Intrinsic pathway

It starts with the activation of Factor XII (Hageman factor) to Factor XIIa with the helpof High molecular weight Kininogen (HMWK) collagen. At the same time, Prekallikrein becomesKallikrein. Factor XIIa then activates Factor XI to XIa which then activates Factor IX to FactorIXa. At the same time Factor VIII, with the help of Thrombin (IIA), activates Factor VIIIa. Factors IXa and VIIIa, together with Calcium, activates Factor X to Factor Xa. Factor Xa activatesFactor II to Factor IIa with the help of activated Factor Va and Calcium. Thrombin (Factor IIa)

activates Fibrinogen, with the help of Calcium, to Fibrin. At the same time, it activates FactorXIII to Factor XIIIa. Finally, Factor XIIIa cross-links Fibrin to form Fibrin Polymer.

Extrinsic Pathway

It starts with the tissue injury with the release of Tissue Factor (Thromboplastin) whichactivates Factor VII to VIIa. Factor VIIa, with the help of Calcium, also activates Factor X to Xa.And the process goes on.. (Final common pathway) Factor Xa activates Factor II to Factor IIawith the help of activated Factor Va and Calcium. Thrombin (Factor IIa) activates Fibrinogen,with the help of Calcium, to Fibrin. At the same time, it activates Factor XIII to Factor XIIIa.Finally, Factor XIIIa cross-links Fibrin to form Fibrin Polymer.


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COAGULATION FACTORS AND FACTOR DEFICIENCIES

Factor I (Fibrinogen) Glycoprotein synthesized by the liver Acute phase reactant

o Increases in response to trauma or to onset of a variety of illnesses Plays the final major step in coagulation

Decreased in severe liver disease Sources: fresh frozen plasma or cryoprecipitate

Factor II (Prothrombin) Proenzyme synthesized by the liver Vitamin K dependent factor Maybe decreased in severe liver diseases Deficiency: Autosomal recessive, extremely rare

Factor III (Tissue Factor)Factor IV (Calcium)

Factor V (Labile Factor) Synthesized by the liver Found in plasma but not in serum Very unstable, must be frozen to maintain activity Deficiency: also called parahemophilia Associated with bruising and soft tissue hemorrhage

Factor VII (Stable Factor) Synthesized by the liver Vitamin K dependent factor

Present in both serum and plasma Deficiency: Autosomal recessive; large hemorrhages into joint, body cavity, intracranial andintramuscular sites; delayed post-surgical bleeding

Factor VIII (von Willebrand Factor complex/Antihemophilic Factor) Found in plasma but not in serum

o Formed by 2 subunits VIII:C (antihemophilic factor)

Synthesized by the liver and is genetically controlled on the X-chromosome

o Factor VIII:R (vWF/ factor VIII-related protein) Synthesized by endothelial cells, megakaryocytes and platelets Has effect on platelet adhesion and platelet aggregation Deficiency: von Willebrand disease

o Classic Hemophilia Sex-limited recessive trait Female carriers and males have clinical diseases Severe bleeding following trauma Bleeding into joints (hemarthrosis), mouth, urinary tract, GIT and

intracranial hemorrhage Depressed factor VIII:C activity; normal vWF; PT and BT are normal; aPTT abnormal

Factor IX (Plasma Thromboplastin Component) X-linked recessive trait Hemophilia B or Christmas Disease Produces a hemophilia-like syndrome Found in plasma or serum Vitamin K dependent factor


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Factor X (Stuart Factor) Present in serum and plasma Part of intrinsic and extrinsic pathways Vitamin K dependent factor

Factor XI (Plasma Thromboplastin Antecedent) Autosomal recessive trait

Present in serum and plasma Mild bleeding

Factor XII (Hageman Factor) Surface contact activator for intrinsic pathway Does not cause clinical bleeding Patients with factor XII deficiency have increased incidence of myocardial infarction and thrombosis

Factor XIII (Fibrin Stabilizing Factor) Autosomal recessive trait Bleeding noted in newborns: umbilical stump bleeding

Also associated with intracranial hemorrhage, soft tissue hemorrhage, recurrent spontaneous abortions,delayed bleeding and poor wound healing

aPTT, PT and BT normal Diagnosis: 5M Urea clot dissolution time

o Measure time it takes for a clot to dissolve in urea o Normal: clot dissolves in 24 hours o In Factor XIII deficiency; clot dissolves faster than 24 hours o

High Molecular Weight Kininogen (HMWK / Fritzgerald Factor) Part of Kallikrein system; affects Factor XII Also plays a role in inflammatory response

Deficiency is rare and no clinical bleeding is induced

Prekallikrein (Fletcher Factor) Deficiency does not predispose to clinical bleeding

LABORATORY EVALUATION OF COAGULATION

Specimen Collection (aPTT and PT)

Whole blood anticoagulated with sodium citrate (citrate binds to calcium and prevents coagulation) 9:1 blood:citrate ratio is required

o A ratio less that 9:1 may falsely increase results Specimen must be assayed as soon as possible and the plasma must be kept cold to avoid factordeterioration

Activated Partial Thromboplastin Time(aPTT)

Reference Value: 25-35s; ratio: < 1.2

Key Facts regarding Coagulation Factors

9 of the 13 coagulation factors are proenzymes that must be activated o Exceptions: Fibrinogen (not an enzyme)

Factor III (tissue factor ) it is a complex rather than a single proteinFactor IV (calcium)

Vitamin K is essential for post-translational carboxylation of factors II, VII, IX, X Many of the coagulation factors are decreased at birth and do not reach adult levels forseveral months. The PT and aPTT cannot be used in newborns

Fibrinogen or platelets are at normal levels at birth, and factor VIII:C and VIII:Ag areelevated at birth

Factor VIII:Ag and Fibrinogen are acute phase reactants (i.e., they are elevated in anycondition associated with inflammation)


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Used in detecting intrinsic pathway abnormalities before prothrombin to thrombin stage Sensitive to effects of heparin Principle: citrated blood is mixed with a negatively charged activator, a partial thromboplastin as aphospholipid source and Calcium. Time to form fibrin clot is measured

Prothrombin Time Reference value: 11-13s; % activity: 70-120; INR:


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o Acquiredo Attack fibrinogenso Eg: in malignancies, extensive surgeries

SECONDARY FIBRINOLYSINSo Naturally occurring; Plasminso Attack fibrin clot to produce FIBRIN SPLIT PRODUCTS

TEST OF FIBRINOLYSINSo Clot lysis test; Fibrinogen assay; thrombin time; PT; APTT; tests for FDP

FIBRINOLYTIC DISORDERS:

Disseminated Intravascular Coagulation (DIC)

Release of tissue thromboplastin and unknown substances liberated into the blood stream and result indeposition of fibrin and fibrin precursor substances like fibrinogen

Major Disorders Associated with DICo Obstetric Complications

Abruptio placentae; retained dead fetus; septic abortion; amniotic fluid embolism;toxemia with pregnancy

o Infections Gram (-) sepsis; meningococcemia; rocky mountain spotted disease; histoplasmosis;

aspergillosis; malariao Neoplasms

Carcinomas of pancreas, prostrate, lung and stomach; acute promyelocytic leukemiao Massive Tissue Injuries

Acute intravascular hemolysis; snakebite; giant hemangioma; shock; heat stroke;vasculitis; aortic aneurysm; liver disease

o S/Sx of bleeding: ARF; RF; tissue hypoperfusion and infarctiono Laboratory tests:

Thrombocytopenia; hyperfibrinogenemia; PT and APTT abnormalities; elevated FDP / D-

Dimer

ANTICOAGULANT SYSTEMS

Coumarin Anticoagulants

Inhibits Vit. K utilization by liver cells affecting Factors II, VII, IX, X Use of coumarin (Warfarin sodium) for antithrombotic activity Monitoring of coumarin therapy by PT Therapeutic range of 1.3 to 1.5 times control value or around 15-20 sec. (N= 11-13 sec.) May also report INR International Normalized Ratio

INR is used for long-term warfarin therapy after patients have been stabilized For standardization of all thromboplastin reagents which permits patients to have comparable results in

any laboratoryHeparin / Antithrombin III (AT-III)

AT-III o Serine protease that binds to and inhibits thrombino Requires heparin as an activator to produce effective anticoagulationo Synthesized by liver

Heparino Biologic substance extracted from porcine gastric mucosa and bovine lung tissueso

Contains a CHO group that binds to AT-III and increase anti-coagulant effect

o Monitoring of heparin therapy by APTT, TTo Therapeutic range is 1.5 2.5 times the APTT upper reference limit

Protein C / Protein S System

Protein Co Serine protease producing inactivation of Factor V and Factor VIII thereby inhibiting conversion

of prothrombin to thrombin o Produced in the liver o Vitamin K dependent


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o Must be activated by THROMBOMODULIN Protein S

o Cofacor needed by Protein C for maximum effect o Synthesized by the liver o Vitamin K depended

Immunologic Type Coagulation Factor Inhibitor

Antiphospholipid antibody (APA) inhibitors of coagulation factor o

Lupus anticoagulant (LAC) o Anticardiolipin Antibody (ACA)

Are of IgG, M or A type antibody type inhibitors of coagulation factors Interfere with coagulation assays that use phospholipids reagents such as APTT

o Effects of thrombosis, fetal death

INVESTIGATION OF A PATIENT WHO IS ACTIVELY BLEEDING

History bleeding / purpura Bleeding time to test for platelet and capillary fragility abnormalities Peripheral blood smear platelet and RBC morphology APTT for intrinsic pathway abnormalities PT for extrinsic pathway abnormalities FDP (D-dimer) for DIC

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CP Blood Coagulation