Charles Eby, M.D.
Saint Louis University Health Sciences Center
October 1999

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  I Outline
1. Overview of hemostasis and fibrinolysis
2. Platelet production and function
3. Quantitative and qualitative platelet disorders
4. von Willebrand factor (vWF) and von Willebrand disease (vWD)
5. Antithrombotic therapies

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  II Response to Vascular Injury
1. Primary hemostasis
1. Endothelial injury exposes flowing blood to subendothelial protein matrix (collagen, vWF, vitronectin, fibronectin, etc.)
2. Platelets adhere to vWF and collagen
3. Platelet activation occurs when exposure to agonists exceeds stimulation threshold
• shape change
• release of granule contents
• aggregation: "platelet plug"
2. Secondary hemostasis
1. Exposure of plasma coagulation factors to tissue factor (TF)
2. Successive activation of coagulation factors terminates in generation of thrombin
1. Thrombin converts fibrinogen to fibrin
2. Fibrin polymerizes, covalently crosslinked by FXIIIa
3. 1⁰ and 2⁰ hemostasis are simultaneous and interdependent processes culminating in formation of a hemostatic plug:
1. Aggregated platelets
2. Fibrin "web"
3. Trapped RBCs, WBC
4. Fibrinolysis
1. tPA activates plasminogen > plasmin
2. Plasmin degrades fibrin into fibrin degradation products
5. Regeneration: Platelet derived growth factors, thrombin, and plasmin participate in repair of subendothelial matrix and repopulation of endothelial cells

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  III Megakaryopoiesis: Committed stem cells proliferate and mature under the control of cytokines and humoral factors terminating in the release of platelets into the peripheral circulation.
1. Committed stem cell stages
• differentiation: endomitosis: 8-64 nuclei
• synthesis of platelet specific membrane receptors and cytoplasmic granules and organelles
• platelet shedding and egress into bone marrow sinusoids
2. Hormones, cytokines and interleukins involved in megakaryopoiesis
• early Thrombopoietin (TPO), IL-3, KL
• mid TPO IL-3; IL-6; IL-11
• late TPO
3. Regulation of circulating platelet mass: current model
• TPO constituetively synthesized liver >> Kidney
• Circulating platelets compete with megakaryocytes for binding free TPO
• ITP: ¯ plt  - free TPO  - stimulation of megakaryopoiesis

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  IV Platelet Structures
1. Granules
• alpha: fibrinogen, vWF, FV, PDGF, platelet factor 4
• dense: ADP, serotonin
2. Cytoplasmic tubules
• open canalicular system: outlet for granule contents
• dense tubular system: Ca++ reservoir
3. Cytoskeleton
• microtubule ring: maintains resting platelet discoid shape
• actin monomers and polymers
4. Platelet adhesion receptors
• GPIb-IX vWF
• GPIIbIIIa fibrinogen
• GPIaIIa collagen
5. Platelet activation receptors:
• ADP, thrombin, epinephrine, serotonin, thromboxane A₂
6. Platelet inhibitor receptors: prostacyclins

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  V Platelet Activation: Most platelet activation receptors are coupled to G proteins which set into motion multiple complex intracellular biochemical processes including:
• "unlocking GPIIbIIIa receptors"
• intracellular [Ca++] secretion of granule contents
• shape changes: disc sphere sea urchin
• arachidonic acid metabolism (cyclooxygenase enzyme dependent) and generation of thromboxane A₂

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  VI Thrombocytopenia
1. Pseudothrombocytopenia
• partial clotting of specimen
• EDTA platelet clumping
• platelet satellitism around white cells
• cold agglutinins
• hereditary macrothrombocytopenia (May-Hegglin)
2. Diminished platelet production
1. Congenital
• Fanconi Syndrome
• Thrombocytopenia Absent Radii syndrome (TAR)
• Wiskott-Aldrich Syndrome
2. Acquired
• Aplastic anemia
• amegakaryocytic anemia
• paroxysmal nocturnal hemoglobinuria (PNH)
• marrow infiltration
• myelophyhistic disease
• granulomatous disease
• storage disease
• radiation
• myelosuppressive drugs
• chemotherapy
• thiazides
• interferon
• ethanol
• nutritional: B₁₂, folate, iron (rare) deficiencies
• HIV
3. Increased platelet destruction
1. Congenital
• intrauterine infections (TORCH)
• Kasabach-Merritt Syndrome
• Alloimmune neonatal thrombocytopenia
2. Acquired
• non-immune
• infections: Ehrlichiosis malaria
• disseminated intravascular, coagulopathy (DIC)
• hemolytic uremic syndrome (HUS) - E coli 0157
• thrombotic thrombocytopenic purpura (TTP)?
• preeclampsia
• HELLP Syndrome: hemolysis, elevated liver enzymes, low platelets
3. Immune mediated: Idiopathic

	Children	Adults
Viral Prodrome	Common	Rare
Self limited illness	Yes	No
Diagnosis	Rule out other causes Bone marrow rarely	Rule out other causes Bone marrow +/-
Treatment	Supportive, IVIG IV Rhogam	Prednisone, splenectomy:  long term remissions IVIG, Rhogam: short term Plt count
Disease Associations	None	SLE

• post transfusion purpura
• rare
• recipient of blood transfusion produces platelet alloantibodies (usu a PLA-1) which behave like platelet autoantibodies
• mechanism unclear
• treatment IVIG
• drug-induced thrombocytopenia
• any drug
• top contenders
• heparin
• Quinine, quinidine
• Vancomycin
• Septra
• HIV
4. Platelet sequestration: "hypersplenism"
• congestive: portal hypertension
• infiltration: lymphoma, myelofibrosis
• storage disease: Gaucher disease
• acute splenic sequestration: sickle cell disease
• granulomas: sarcoidosis: Tb
5. Acute blood loss

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  VII Thrombocytosis
1. Primary
1. myeloproliferative disease
• essential thrombocythemia
• polycythemia vera
• CML
• agnogenic myelofibrosis
2. Myelodysplastic syndromes
• 5q- syndrome
• RARS
3. Potential complications: thrombotic (erythromelalgia) and hemorrhagic
2. Secondary (reactive)
• infectious/inflammatory conditions
• malignancy
• iron deficiency anemia
• splenectomy

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  VIII Qualitative Platelet Disorders
1. Clinical presentation
• petechiae and ecchymoses
• mucosal bleeding
• menorrhagia
• immediate excessive bleeding post trauma
2. Laboratory evaluation of platelet function
1. bleeding time (BT)
1. poor precision and accuracy due to:
1. poor operator technique
2. patient variables
• medications
• cooperation
• skin condition
2. Major misconceptions regarding BT results:
• BT is an in-vivo test of platelet function
• BT is a sensitive and specific predictor of increased bleeding prior to or after surgery in unselected patients.
• BT can be correctly performed by anyone
• There is a strong correlation between BT and thrombocytopenia
3. Indication for BT:
1. evaluation of subjects with personal and/or family histories of prolonged bleeding and easy bruising
2. In-vitro platelet aggregation studies
• monitor change in light transmission through patient's platelet rich plasma after adding different agonists (ADP, collagen, arachidonic acid) or ristocetin, an antibiotic that agglutinates platelets via vWF binding to GPIb-IX receptors.
3. Congenital qualitative platelet disorders
1. Adhesion
• Bernard-Souliers Syndrome: Rare, autosomal recessive, GPIb-IX receptors absent, platelet adhesion to vWF severely impaired, mild thrombocytopenia and larger platelets
• Glanzmann's thrombasthenia: rare autosomal recessive, GPIIbIIIa receptors absent, platelet aggregation severely impaired. Platelet morphology normal
• Pseudo-vWD: vWf binds to resting platelets due to abnormal GPIb-IX receptors can be confused with type 2B vWD
2. Secretion
• Dense granule deficiencies (d storage pool deficiency)
• autosomal dominant disorder, nl plt count and morphology, or associated with other congenital defects
• Hermansky-Pudlak
• Chédiak-Higashi
• TAR
• Wiskott-Aldrich Syndrome
• Gray platelet syndrome: autosomal recessive absence of a granules
3. Activation: common, heterogeneous
• defects in arachidonic acid metabolism and thromboxane A₂ receptor
• defects in signal transduction pathways

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  IX Acquired qualitative platelet disorders
• uremia
• post-cardiopulmonary bypass surgery
• liver disease
• dysproteinemias
• myeloproliferative disorders
• Cox-1 enzyme inhibitors (aspirin, NSAIDS)
• other drugs: B-lactam antibiotics, ETOH, dextran

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  X von Willebrand disease (vWD): Clinical syndrome due to quantitative and qualitative disorders of vWF
1. vWF Structure
• gene expressed in megakaryocytes and endothelial cells
• dimerization of transcribed 250,000 mw propeptide occurs in endoplasmic reticulum
• N-terminal disulfide linkages between dimers produces a range of large vWF multimers (up to 1-20 x 10⁶ mw)
• vWF is stored in platelet a granules and Weibel-Palade bodies in endothelial cells
• discrete domains are associated with specific functions
2. vWF Functions
• platelet adhesion to subendothelial matrix via GPIb IX receptors
• platelet aggregation via GPIIbIIIa receptors
• stabilization of FVIII
3. vWD
• 1926 Eric von Willebrand described family from a Finnish island with autosomal dominant inherited bleeding disorder. Bleeding times were prolonged and FVIII activities low in affected subjects.
• Now recognized as most common inherited bleeding disorder, frequency ~1%, but penetrance is incomplete, and diagnoses are based on a battery of hemostasis tests which lack sensitivity and specificity.
4. Panel of Tests Used to Screen for vWD
• Bleeding time: often mildly prolonged due to abnormal platelet adhesion but nl BT has poor negative predictive value.
• Factor VIII activity usually decreased due to decreased vWF Ag or abnormal FVIII binding region
• vWF antigen: concentration of vWF protein
• Ristocetin cofactor activity vWF:RCo. An in vitro test to assess patient vWF adhesion of preserved donor platelets in the presence of ristocetin, an antibiotic introduced in the 1950's and quickly withdrawn due to severe thrombocytopenia
5. Tests for Subtyping vWF
• RIPA (ristocetin-induced platelet agglutination). The degree of platelet agglutination is monitored when low and high concentrations of ristocetin are added to patient's platelet rich plasma.
• vWF multimer analysis: electrophoresis of plasma sample on 1-2% agarose gel, transfer to nitrocellulose, vWF multimers visualized with radiolabeled or enzyme conjugated anti-vWF antibody
6. Classification of vWD

	Type 1	Type 2A 2B 2N	Type 3
Relative frequency	70%	------------- 30% ---------------	< 1%
Inheritance	auto dom.	auto auto auto dom. dom recessive	auto recessive
Severity	mild>moderate	------------ mild<moderate --------	severe
Therapy	DDAVP vWF conc.	DDAVP no DDAVP DDAVP -----------vWF concentrate --------	vWF concentrate
FVIII:C	15-50%	15-50% 15-90% 1-30%	<10%
vWF:Ag	15-50%	¯ ¯/nl nl/¯	<5%
RCO	15-50%	¯¯ ¯¯ nl/-	<5%
vWF multimers	nl	loss of large bands nl	absent
RIPA	¯	¯ - nl/¯	absent

7. Type 1 vWD diagnostic challenges
1. lab test results over lap with lower end of reference range due to:
• hormone therapy
• pregnancy
• acute illness
• difficult phlebotomy
2. imprecision of bleeding time, vWF:RCo
3. influence of ABO on reference ranges for FVIII, vWF:Ag, vWF:RCo
4. incomplete penetrance of bleeding tendency in families
5. genetic diagnosis of type 1 vWD not yet feasible
8. Management of vWD
• temporarily increase circulating vWF by stimulating release of endothelial cell stores with DDAVP
• analog of vasopressin
• parenteral (IV, SQ, nasal)
• passively infuse vWF
• 1^st choice Humate-P: a Factor VIII concentrate rich in vWF
• 2^nd choice cryoprecipitate
9. Current model: stimulation of a procoagulant response results from a complex interaction between cellular, procoagulant, and anticoagulant components that exceeds a limiting threshold. Once initiated, activated platelet phospholipid membranes provide a surface for coagulation enzyme and cofactor complexes to organize, leading to an explosive generation of thrombin which simultaneously promotes and inhibits clot formation, and initiates clot lysis and vascular repair.

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  XI Routine Coagulation Tests
1. Preanalytical variables
1. may use first venipuncture specimen
2. 3.2% Na citrate recommended vs 3.8%
3. hct >55 may give false APTT, PT elevations
4. inadequate filling (<90%) may give false APTT, PT elevations
5. incomplete removal of platelets may shorten APTT, PT
2. Activated Partial Thromboplastin Time (APTT)
• 2 steps
• assesses intrinsic and common pathways
3. Prothrombin Time (PT)
• 1 step
• assesses extrinsic and common pathways
4. Thrombin Time (TT)
• 1 step
• assesses thrombin inhibitors, quantitative and qualitative fibrinogen defects
5. Fibrinogen: Clauss method
• functional, expressed as ng/dl
• plasma diluted 1:9, thrombin cone 10 x TT
• insensitive to thrombin inhibitors
6. Coagulation Factor Activities
• PT (FVIII, X, V, prothrombin) or APTT (intrinsic factors) bases
• plot clotting times of 50:50 mixture of deficient plasma and dilutions of reference plasma (pooled normal plasma, all factors 100% activity) on logrithmic scales: linear
• perform clotting time on 50:50 mixture of deficient plasma and patient plasma
• determine % activity from standard curve
7. Coagulation Factors
• FXIII (fibrinogen) synthesized in liver
• serine protease proenzymes: FXII, Prekallikrein, XI, X, IX, VII, prothrombin
• vitamin K dependent gamma carboxylation: FX, IX, VII, prothrombin (Pro C, Pro S)
• vitamin K dependent factors are low (related to adults) in neonates until 6-12 months

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  XII Hemophilias
1. Hemophilia A: X-linked inheritance ~10/100,000 males - APTT, nl PT
1. Severe <1% FVIII:C
• frequent, spontaneous hemarthroses, muscle hematomas
• 10-20% develop neutralizing FVIII antibodies (inhibitors)
2. Moderate 1-5% FVIII:C
• spontaneous bleeding less frequent than severe
• inhibitors rare
3. Mild >5-30% FVIII:C
• excessive bleeding with trauma
4. Symptomatic women
• carrier with low FVIII:C (lyonization)
• Turner XO carrier
• inherited two hemophilia X chromosomes
5. Carrier status determination
• FVIII:C ¸ vWF:Ag: historical
• RFLP: most informative in moderate, mild forms
• direct gene mutation analysis for intron 22 inversion (50% severe)
6. laboratory diagnosis of FVIII inhibitor
• bleeding not controlled, APTT not shorter after FVIII infusion
• 50:50 mix does not normalize APTT; time dependent
• quantitative inhibitor strength: Bethesda Unit
• reciprocal of patient plasma dilution that neutralizes 50% of FVIII:C in PNP after 2^o incubation 37^o C.
7. treatment
• mild hemophilia A, minor trauma: DDAVP and Amicar (fibrinolysis inhibitor)
• moderate/severe hemophilia A or major trauma in mild hemophilia A
• recombinant FVIII
• monoclonal purified plasma FVIII
• 1u/kg = 2% rise in plasma FVIII:C, t ½ 12 h
• inhibitor patients
• 1^st porcine FVIII
• 2^nd recombinant FVIIa
• 3^rd activated prothrombin complex concentrate (FEIBA)
2. Hemophilia B: X-linked ~3/100,000 males -APTT, nl PT
1. severity of bleeding proportional to FIX:C
2. bleeding complications identical to hemophilia A
3. inhibitors less common ~3%
4. treatment
• recombinant FIX
• monoclonal purified plasma FIX
• 1u/kg = 1% rise plasma FIX:C, t ½ 24 h
• inhibitor patients
• 1^st rFVIIa
• 2^nd FEIBA

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  XIII Other Congenital Factor Deficiencies
1. Factor concentrates unavailable. Use FFP or Cryoppt (Fibrinogen, FXIII)
2. FXII, Prekallikrein, Heavy Molecular Weight Kininogen
• auto recessive, markedly - APTT, nl PT
• no abnormal bleeding
• no factor replacement necessary
3. FXI
• auto recessive, - APTT, nl PT
• Ashkenazi Jewish ancestry
• bleeding less severe than hemophilia A/B
4. FVII
• auto recessive, nl APTT, - PT
• severe bleeding <1%
• minimal bleeding >10%
5. FX, V, Prothrombin, afibrinogenemias
• auto recessive, very rare, - APTT and PT
6. FXIII: Auto recessive nl APTT and PT
• clinically important if <1%
• bleeding: umbilical stump, trauma, spont. CNS
• spontaneous abortions
• poor wound healing
• laboratory diagnosis
• immunoassay
• stability of fibrin clot in 5 m urea
• replacement: cryoppt
7. a₁ antitrypsin Pittsburgh
• point mutation cause ATIII activity
• accelerated thrombin inhibitor

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  XIV Acquired Coagulation Factor Deficiencies
1. Vitamin K and deficiencies of FX, IX, VII, prothrombin
• PT prolonged >APTT
• nutritional deficiency
• competition (warfarin)
2. Liver disease
• all factors except FVIII, vWF can be decreased
• Vit K dependent factors and FV more sensitive
3. Amyloidosis
• absorption of FX to amyloid increases clearance
4. Factor specific autoantibodies, FVIII most common (1-2/million/yr)
• patients usually >65
• 50% idiopathic, 50% associated with underlying diseases: RA, SLE, lymphoma, autoimmune skin diseases, post partum
• presentation: sudden onset of bleeding and bruising. - APTT, PT normal, APTT mixing study prolonged, ¯ FVIII:C
• management: treat severe bleeding with porcine FVIII
• immunosuppression with prednisone, +/- cytoxan is usually successful if patient does not bleed to death first!

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  XV Regulators of Coagulation
1. Antithrombin III
• member of serine protease inhibitor (SERPIN) family
• covalently binds to active sites of FXa and thrombin
• accelerated by endogenous heparinoids on endothelial membrane
• accelerated by exogenous heparin
• supportive role: a₂ macroglobulin, heparin cofactor II
2. Protein C
• vitamin K dependent serine protease
• activated to APC by thrombomodulin bound thrombin
• proteolyses FVIIIa and FVa to stop thrombin generation
3. Protein S
• vitamin K dependent, but no enzymatic activity
• free (40%) vs C4_b binding protein bound (60%) equilibrium
4. Tissue factor pathway inhibitor (TFPI)
• direct inhibition of FXa
• FXa-TFPI complex inhibits tissue factor-FVIIa
• prevents minor tissue injuries from generating thrombin gate keeper
• thrombotic event due to congenital deficiency not confirmed.

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  XVI Inherited Risk Factors for Recurrent and Spontaneous Various Thromboembolic Events (VTE)
1. Current model: VTEs are a consequence of polygenetic risk factors combined with acquired, usually temporary, acquired risk factors:
• trauma/surgery
• pregnancy/OCPs/ERT
• MI/infection/inflammatory disease
• immobility and trauma
• antiphospholipid antibodies
2. The impact of undiscovered genetic risk factors cannot be measured, leading to overweighing the VTE risk associated with known factors.
3. Inherited deficiencies of coagulation regulators
1. partial deficiencies (autosomal dominant, incomplete penetrance)

Regulator	Population Freq.	VTE pop. Freq.	Acquired deficiency
Protein C	1:300	1-5%	LD, VK, War, DIC
Protein S	? est. 1:300	1-5%	LD, VK, War, DIC
Antithrombin III	1:2000	1-5%	LD, heparin, DIC
L.D. = liver disease, VK = vitamin K deficiency, War = warfarin

2. Homozygous deficiencies (very rare)
1. Protein C and S
• neonatal purpura fulminans
• warfarin associated skin necrosis
2. ATIII
• total deficiency never reported
• incompatible with fetal life?
4. D. Coagulation factor mutations associated with increased VTE risk
1. Factor V Leiden (FVL) discovered 1994
• point mutation substitutes gln for Arg 506
• eliminates 1 of 3 APC cleavage sites
• slower inactivation of FVa, more thrombin generation
• functional assay to screen for FVL: Activated Protein C ratio
• positive if APTT + APC ¸ APTT <2.0
• frequency of FVL among races
• whites ~5-7%
• Hispanics ~3%
• African Americans ~2%
• Asians <1%
• frequency among patients with VTE, 20-60%
2. Prothrombin 20210 point mutation
• located downstream of exons coding for prothrombin (3~ region)
• associated with higher prothrombin activities
• frequency about ½ of FVL
• relative risk for VTE similar to FVL
5. E Metabolic risk factors for VTE: hyperhomocysteinemia (HHC)
• consequence of defective methionine metabolism
• epidemiologic data associates HHC with arterial and venous thrombi
• disease mechanisms unknown - focus is on vessel wall
• vitamin supplementation (folate, B₆, B₁₂) can lower HC
• benefit unproven at this time

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  XVII Antiphospholipid antibodies (APA)
1. APAs are a heterogeneous group of autoantibodies that recognize certain proteins associated with phospholipid membranes. Clinical and biochemical characterization of these antibodies is incomplete and prediction of outcomes for asymptomatic patients with APAs is difficult.
2. Laboratory detection
1. interference with phospholipid-based clotting tests: Lupus anticoagulant (LA)
• in general APTT is more sensitive than PT to LA
• many assays, no accepted "gold standard"
• dilute Russell Viper Venom Test (dRVVT)
• STA CLOT LAâ
• kaolin clotting time
• dilute thromboplastin time
• platelet neutralization of prolonged APTT
2. minimal criteria for LA testing
• perform two or more sensitive screening tests
• if screen test - r/o factor deficiency by repeating on 50:50 mix
• if still -, confirm clotting time ¯ after adding more phospholipid
• rule out specific coagulation factor inhibitor (FVIII!)
3. binding of patient immunoglobulins to solid phase phospholipid
• cardiolipin most frequently used phospholipid
• epitope is B₂ glycoprotein 1 - cardiolipin complex
• conjugated a human IgG, IgM, IgA antibodies detect anticardiolipin antibodies
3. APA associations
1. idiopathic "benign": unexpected pre-operative prolonged APTT
2. reactive and frequently fleeting: drugs, infections, inflammation, malignancy
3. presumed to be part of pathogenesis of the following complications in association with SLE (secondary) or idiopathic (primary phospholipid antibody syndrome)
• venous and arterial thromboembolic events
• recurrent spontaneous abortions
• thrombocytopenia

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  XVIII Disseminated Intravascular Coagulopathy (DIC)
1. A complex syndrome complicating many acute and chronic diseases. The essence is unregulated thrombin generation and reactive plasmin activity. A diagnosis of DIC requires combining serial clinical and laboratory findings.
2. Causes of excess thrombin generation
• exposure of blood to excess tissue factor: trauma, surgery, placental complications, malignancies, some snake bites
• endothelial injury: burns, sepsis, venoms, A-V malformations
• procoagulants: snake venoms, acute promyelocytic leukemia (APL), other solid tumors
• fibrinolytic activity: APL, rarely, solid malignancies, snake venoms
3. DIC presentations
1. acute: seriously ill patients with signs and symptoms of:
• micro thrombi: skin necrosis, acrocyanosis, global organ dysfunction: CNS, respiratory, renal
• bleeding from multiple sites
• skin: venipuncture, purpura
• mucous membranes
• GI
• CNS
• Laboratory assessment of acute DIC: no single test is diagnostic for DIC. Instead, the results of a panel of simple, rapid, expensive tests are combined with the clinical status of the patient.
• platelet count usually < 150,000
• APTT, PT may be prolonged
• fibrinogen lower than expected
• D-Dimer* elevated
• peripheral smear schistocytes (insensitive)
• * D-dimer is an immunoassay that detects a unique fibrin degradation product: the D-domains of two fibrin molecules that have been covalently linked by FXIIIa. An elevated D-dimer always indicates plasmin activity, but it may not be part of DIC. D-dimers are elevated after surgery, trauma, and VTEs.
2. Chronic DIC: typically patients with:
• GI adenocarcinomas
• venous thromboembolic events
• recurrent migratory superficial phlebitis (Trouseau's Syndrome)
• laboratory findings:
• compensated consumption
• normal plt count, fibrinogen, PT
• APTT may be abnormally short (?circulating thrombin?)
• D-Dimer elevated

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  XIX Monitoring Anticoagulation Therapy
1. Acute, parenteral anticoagulation with heparin
• purified from porcine/bovine intestines/lungs
• variable lengths of sulfated polysaccharides
• accelerates ATIII inhibition of FXa and thrombin
• therapeutic range empirically determined
• old: 1.5 - 2.5 x normal APTT
• new: APTT range corresponding to 0.3 - 0.6 units of heparin/ml
• low molecular weight heparins: SQ delivery, weight based dosing, no laboratory monitoring required
• heparin induced thrombocytopenia (HIT)
• 1-3% of patients exposed to heparin
• antibodies (IgG recognize heparin-platelet factor 4 complexes
• IgG-heparin-PF4 bind to platelet Fc receptor > platelet activation
• acquired hypercoagulable state frequently complicated by:
• DVT, PE, arterial thromboembolisms
• alternative anticoagulants: heparinoid, recombinant hirudin
2. Chronic oral anticoagulation with warfarin (Coumadin)
• competitive inhibitor of vitamin K dependent gamma carboxylation in hepatocytes
• decreases FX, IX, VII, prothrombin, Protein C, Protein S activities
• variation in PT reagent (thromboplastin) sensitivities to warfarin effect lead to development of ISI system:
• human brain thromboplastin (TP) chosen as WHO reference
• commercial thromboplastin protimes compared to WHO standard protimes for controls and patients on warfarin
• ISI = 1 if identical
• ISI <1 if commercial TP more sensitive to warfarin effect
• ISI >1 if commercial TP is less sensitive to warfarin effect

INR = (PT patient on warfarin)ISI ( mean reference PT )

• ideally, INR results from different laboratories performed on same plasma specimen should be identical.
• ISI calculation errors and specific instrument variables cause of most INR disparities
• therapeutic range INR 2-3 for DVT, PE, chronic atrial fibrillation.

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  XX Fibrinolysis: The proteolysis of fibrin by the serine protease plasmin is a highly regulated process restricted to the fibrin clot.
1. Components
1. tissue plasminogen activator (tPA)
• serine protease
• synthesized by endothelial cells
• binds to and inhibits fibrin and activates plasminogen
2. plasminogen activator inhibitors (PAI-1)
• member of Serpin family
• synthesized by hepatocytes, platelets, endothelial cells
• covently binds to plasma tPA
3. plasminogen
• Serine protease
• activated by tPA
• binds to lysine residues on fibrin
• capable of proteolyzing fibrin, fibrinogen, FVa, FVIIIa, platelet glycoproteins
4. a₂ antiplasmin
• member Serpin family
• covalently binds to and inhibits free plasmin
2. Laboratory monitoring of fibrinolysis
1. Euglobulin lysis time
• global in-vitro test of fibrinolytic activity
• dilute, acidified plasma: plasminogen, tPA, plasmin, fibrinogen PPT
• add thrombin, form fibrin clot
• clot lysis time <90 min = hyperfibrinolysis
2. Plasminogen
• antigenic and functional assays
• congenital hypo plasminogenemia, weakly associated with DVT/PE
3. tPA, PAI-1, a₂ AP
• antigenic and functional assays
• not routine tests
3. Inherited hyperfibrinolysis states (auto recessive): post trauma/surgery bleeding
• PAI-1 deficiency
• a₂ antiplasmin deficiency
4. Acquired hyperfibrinolysis
• liver disease
• acute promyelocytic leukemia
• thrombolytic therapy: acute MI, CVA

1 Schiffman FJ (editor). Hematologic Pathyphysiology. Lippincott-Raven, Philadelphia, 1998

2 Henry JB (editor). Chapters 28 (Blood Platelets) and 29 (Coagulation and Fibrinolysis). In Clinical Diagnosis and Management by Laboratory Methods, 19^th Edition. W.B. Sanders, Philadelphia, 1996, pp. 701-747