HEMOSTASIS 2

Dr. Suzana Voiculescu,

Assist. Prof., Discipline of Physiology and Fundamental Neuroscience

REVIEW- CLASSIC VS MODERN

CELL BASED MODEL OF HEMOSTASIS

Specific cellular receptors for coagulation

proteins promote hemostasis which occurs

in three overlapping phases

Initiation – involves tissue-factor bearing cell

and production of small amounts of thrombin

Amplification – involves platelet activation and

sets the stage for large scale thrombin

production

Propagation – the activated platelet surface

protects factor XI and results in an explosive

burst of thrombin formation

CELL BASED MODEL OF HEMOSTASIS

NATURAL ANTICOAGULANT SYSTEM

PROTEIN C

62-kD vitamin K-dependent glycoprotein

Synthesized in the liver as a single-chain

zymogen

Clipped into a serine-protease-like enzyme on

phospholipid cell surfaces by thrombin

PROTEIN C

Activated protein C (APC) binds protein S for

further activity

Protein C also has pro-fibrinolytic, anti-

inflammatory and anti-ischemic properties

PROTEIN S

Require negatively charged phospholipids and

Ca2+ for normal anti-coagulant activity

Complexes with protein C and acts by

proteolyzing Factor VIII and Factor V, which in

turn prevents activation of factor X and

prothrombin

PROTEIN S AND C DEFIECENCY

Genetic- homo/heterozygous

Acquired

Liver disease

DIC

Vitamin K deficiency

Septic shock

CLINICAL

Deep vein thrombosis is the most common

Pulmonary embolism

Fetal loss

THROMBOMODULIN

specific endothelial cell receptor that forms a

1:1 stoichiometric complex with thrombin

Cofactor in the thrombin-induced activation of

protein C

responsible for the conversion of protein C to

the activated protein C .

ANTICOAGULANT THERAPY

Injectable- heparin and heparin derivatives

Unfractioned heparin

Low molecular weight heparins

Oral- antivitamin K inhibitors

Coumadins- most used= WARFARIN

HEPARIN AND HEPARAN SULFATE

Heparan sulfate- is a GAG

Similar structure to heparin

Endogenous heparin- secretory granules of mast cells (probably being negatively charged- retains histamine inside)

Similar synthetic pathways

Function of heparan sulfate in coagulation- anticoagulant- receptor for ATIII activation thrombin inhibition

HEPARAN SULPHATE

vast structural diversity

bind and interact with a wide variety of

proteins, such as growth factors, chemokines,

morphogens, extracellular matrix components,

and enzymes

modulate different biological processes

TYPES OF HEPARIN

Unfractioned- high molecular weight; injectable

iv, hospitalised patient usually, APTT monitoring

needed (administred using a heparin pump)

Low molecular weight heparin- subcutaneously,

home therapy, no APTT monitoring, less

bleeding as side effect

UNFRACTIONED HEPARIN AS A DRUG

Needs ATIII as a cofactor

Inhibits Xa and thrombin

Clinical uses

Venous thrombosis disorders

Pulmonary embolism

Prophylaxis and treatment- thrombosis in major surgeries

Extracorporeal circulation

Blood samples drowned for lab purpuses- AC

Blood transfusions- in vitro AC

LOW MOLECULAR WEIGHT HEPARIN USE

No APTT monitoring needed

Lower molecular weight

May be administrated subcutaneously- may be

used for unhospitalized patients (once/twice a

day)

Less bleeding

Tends to replace heparin in venous thrombosis,

pulm embolism, acute coronary syndomes

ADVANTAGES OF LMWH OVER UH

Less bleeding (less inhibition on platelet

function)

Longer plasma half life (4-6 over 1 h)

No monitoring for LMWH

Less incidence of thrombocytopenia

HEPARIN TREATMENT COMPLICATIONS

Haemorrhage

Thrombocytopenia (most common drug-

induced thrombocytopenia)

Type I- nonimmune (2 days after treat)

Type II- immune (4-10 days after- HIT)

Osteoporosis (when long- term admin)

ORAL ANTICOAGULANT THERAPY

Vitamin K competitors- coumadin (Warfarin)

Warfarin inhibits the vitamin K-dependent

synthesis of biologically active forms of the

calcium-dependent clotting factors II, VII, IX and

X, as well as the regulatory factors protein C,

protein S.

VITAMIN K DEPENDENT FACTORS

VITAMIN K

Lipidic vitamin- diet (green veg and oils); colic

bacteria synthesis

Haemostasis

Bone matrix proteins, osteocalcin, undergo

gamma carboxylation with calcium much the

way coagulation factors do; needs VK

VITAMIN K DEF

according to the age

most often in infancy (lack of VK reaching the fetus across the placenta, the low level of VK in breast milk, and low colonic bacterial synthesis)

large amount of VK given to a pregnant patient can lead to jaundice in a newborn

Adults- 1 week reserve, abs in terminal ileum in lymph (bile salts, microvilli)

VITAMIN K (VK)

Cofactor

needed for the conversion of 10-12 glutamic

acid residue on the NH2 -terminal of precursor

coagulation proteins into the action form of

gamma-carboxyglutamic acid

VK-dependent gamma-glutamyl carboxylase

allows the VK-dependent proteins to bind to

surface phospholipids

In the liver- vitamin K helps the carboxylation of glutamic acid residues on immature coag factors; in this process it gets oxidized

Antivitamin K drugs inhibit reductase by inhibition, vitamin K stays in an inactive form

ANTI VITAMIN K

Anticoagulant effect starts in 24-36 h from

initiation – stops in 36-72 h after it is stopped

Oral treatment

At first- injectable treatment overlaps the oral

one (at least 4 days)

ANTIVITAMIN K CLINICAL USES

Venous thrombosis

Stroke

Thrombembolism

Cardiac valve replacement

Post myocardial infarction

MONITORING HEMOSTASIS

Primary

Bleeding time

Rumpel Leede

PLT count

Secondary

Clotting time

Howell Gram time

APTT

PT (Quick time)

HOWELL GRAM INTRINSIC AND COMMON

test explores the intrinsic and common pathways; for this we add CaCl2 to citrate plasma and start monitoring the time needed to clot.

Normal values= 60 -120s

High:

TR disfunction / thrombocytopenia

intrinsic pathway factor deficit (XII,XI,IX,VIII- hemophilia)

common pathway deficit- X, V, II, I (hypofibrinogenemia/ afibrinogenemia)

-anti- clotting therapy- heparin

APTT ACTIVATED PARTIAL THROMBOPLASTIN

TIME INTRINSIC AND COMMON PATHWAYS

To citrate plasma (with low amount of platelets- centrifuge for 15 min- 5000 rpm) we add Ca Cl2, kaolin, cephalin and start monitoring the time needed to clot.

Cephalin is a partial tromboplastin (only phospholipid)

Kaolin or silica is a negatively charged molecule activates factor XII

Normal values = 30- 40 s

Causes of abnormal high values- the same as Howell- except for the platelet- derived causes

It is affected by unfractioned heparin- used to monitor therapy

APTT

APTT INTERPRETATION- PROLONGATION

Deficiencies of XII,XI,IX,VIII (but in mild def is normal- the def factor needs to be <20-40% for aPTT to be low)

Antibodies against fVIII (aquired hemofilia) or lupus anticoagulant present

Liver disease

Unfractioned heparin use

APTT

May also be affected by:

Coumarin high dosage therapy

Lupus anticoagulant presence- antibody with in

vivo prothrmobotic activity, but in vitro

antithrombotic activity (interferes with the PL

used in anticoagulat tests procedure)

PROTHROMBIN TIME (QUICK METHOD-PT/QT)

EXTRINSIC AND COMMON

To citrate plasma (with high amount of

platelets) we add Ca Cl2, thromboplastin and

start monitoring the time needed to clot.

Elevated in

Deficit of I, II, V, VII, X factors

Liver failure

K vitamin deficiency/ anti vit K anticoag therapy

Normal values = 12-15 s

PROTHROMBIN TIME

Used to monitor therapy with oral anticoagulant drugs- vitamin K blocking agents- coumarins (warfarin)!!!

By INR fraction which should be measured constantly in these patients

The therapy is used in patients with high thrombotic risk

INR

International normalized ratio

ISI- depends on the tissue factor, it is established

by the manufacturer (usually between 1- 2)

Normal range for a healthy person= 0.9- 1.3

Warfarin therapy monitoring- depends on

pathology

THROMBIN TEST

Explores the last coagulation step, except for factor XIII

Thrombin is added to the plasma directly time needed for clot to form

Normal values- 15-18 s

High in:

Heparin therapy

Abnormal fibrinogen (qualitative and quantitative)

Lupus anticoagulant present

HEMOPHILIA

group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation

Excessive bleeding, minor injury

Sex- linked

Type A- VIII factor deficit

Type B- IX factor deficit

Not sex- linked

Type C- XI factor deficit

Severity varies due to level of active clotting factor (<1 %- severe)

FIBRINOLYSIS

As the clots forms, it incorporates plasmin molecules.

Plasmin is an enzyme formed from plasminogen by tissue plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA)

Plasmin breaks down fibrin polymers into fibrin fragments = fibrinolysis

Fibrinolysis helps removing the clot as the repair processes occur.

FIBRINOLYSIS

Damage to the tissues releases TPA, which together with activated components from the coagulation pathways and protein C, activates plasminogen to plasmin.

Plasmin acts on the insoluble fibrin to form a series of soluble products: FDPs.

Fibrin that has been stabilized (crosslinked) by factor XIII gives rise to crosslinked FDPs (XDPs), as well as X Y D and E fragments.

The XDPs (D-dimer, D-dimer-E fragments), and oligomers of fragments X and Y, can be detected using antibody coated latex beads.

PLASMINOGEN

Zymogen plasmin

GP

Syntesized in liver

Lyses fibrin into fibrin degradation products (FDP)

PHYSIOLOGICAL ACTIVATORS OF PLASMIN

tPA- released from endoth cells responsable

for intravascular fibrinolysis- mainly activates

plasminogen bound to fibrin

uPA- high conc in urine responsible for

extravascular fibrinolysis

PHYSIOLOGICAL INHIBITORS OF PLASMIN

PAI-1 is the physiological plasminogen

activatior inhibitor 1- (tPA)- from platelets

Alpha 2 antitripsin- inhibits plasmin directly

FIBRINOLYTIC THERAPY= THROMBOLYTIC

Used to dissolve blood clots- act by

plasminogen activation

3 major classes:

tPA used in miocardial infarction therapy

uPA

sPA (streptokinase activator)

HEMOSTATIC BALLANCE

The regulation of hemostatic and fibrinolytic processes is dynamic Balance between

Pro- and anti-hemostatic mediators

Pro- and anti-fibrinolytic mediators

Balance can be upset if any components are Inadequate

Excessive

Development of thrombi Excessive local or systemic activation of coagulation

Sustained bleeding Excessive local or systemic fibrinolytic activity

When hemostasis is delayed Either platelet disorder or a coagulation defect

Bleeding episode may be prolonged

Imbalance created between An abnormally slow hemostatic rate

A normal rate of fibrinolysis

An inadequate fibrinolytic response May retard lysis of a thrombus and even contribute

to its extension

CLINICAL CASE 1

23 year old male.

Over the past week noted increasing fatigue,

sore throat, earaches, headaches, and episodic

fever and chills. Unable to run his customary 25

miles per week.

Erythematous throat and tonsils.

Swollen cervical lymph nodes.

DIAGNOSIS INFECTIOUS MONONUCLEOSIS

“kissing disease”

Viral- Ebstein Barr virus

90% asymptomatic

Pharyngitis, fatigue, malaise, fever

CASE STUDY 2

70 year old female.

Symptoms of dyspnea on exertion, easy

fatigability for past 2 to 3 months.

Physical exam- palor

BLOOD SMEAR

Morphologic Alterations Results of the blood smear exam were:

RBC morphology: 2+ hypochromasia 3+ microcytosis 2+ anisocytosis 2+ elliptocytes and target cells occ teardrops and fragments

WBC morphology: Within normal limits

(one lymphocyte shown here)

PLT morphology: Within normal limits

Iron studies were performed, and results were:

serum ferritin <10 ng/mL (RI 12-86)

serum iron 24 µg/dL (RI 65-175)

TIBC 729 µg/dL (RI 250-410) saturation 3 % (RI 20-55)

DIAGNOSIS- IRON DEFICIENCY ANEMIA

Clinical Course

Diagnostic procedures included upper GI endoscopy, colonoscopy,

and small bowel biopsy. All were negative.

The patient received packed RBC transfusions and was started on iron therapy.

The etiology of her iron deficiency anemia could not be determined,

but it was most likely nutritional.

CASE STUDY 3

25 year old male.

Recurrent upper respiratory infections with

fever, nausea, and submandibular swelling for

several months prior to admission.

Noted that cuts on his hands did not heal well.

Physical Exam

Submandibular adenopathy.

No other organomegaly.

RBC morphology: Normochromic 1+ polychromasia 1+ macrocytosis

WBC morphology: The abnormal cells are medium-sized blasts. The nuclei are often irregular in shape, and some have invaginations or deep clefts.

Most have a fine chromatin pattern and one or more prominent nucleoli.

The cytoplasm is basophilic, and thin Auer rods are seen.

PLT morphology: Within normal limits

FURTHER LABORATORY STUDIES BONE

MARROW BIOPSY:

Aspirate:

The differential showed 93.6% blasts similar to

those in the blood.

DIAGNOSIS ACUTE MYELOBLASTIC LEUKEMIA

Chemotherapy was started, and remission was induced within one month.

The post-induction marrow showed normal regeneration, with 2.5% blasts.

The patient was placed on consolidation chemotherapy, and followed in Oncology Clinic.

He was still in remission 4 years after diagnosis.

Note: The patient's MCV returned to the normal

range shortly after induction was started, and the cause of his mild macrocytosis was not investigated.

CASE STUDY 4

40 year old female.

Brought to Emergency Room with symptoms of severe frontal headache and associated confusion. Noted to have decreased energy level and a 15 pound weight loss over the previous three months.

Physical Exam

Pale appearing, but otherwise within normal limits. No organomegaly.

SMEAR

RBC morphology: Normochromic 2+ anisocytosis 2+ oval macrocytes 1+ teardrop cells

WBC morphology: Dysgranulopoietic changes, including hyposegmentation, pseudo-Pelger-Huët nuclei, and hypogranularity are seen in some neutrophils (one shown here).

PLT morphology: Within normal limits

CASE STUDY 5

34 year old female. Two day history of

ecchymoses, petechiae, and hematuria. She

had noted headaches, nausea, and increasing

dysphoria over the past week.

Physical Exam: Mild scleral icterus. Scattered

ecchymoses and petechiae. Appeared anxious

and agitated.

SMEAR

RBC morphology: normochromic 2+ polychromasia 3+ anisocytosis 3+ fragments 2+ spherocytes

WBC morphology: Within normal limits (one lymphocyte shown here) PLT morphology: Within normal limits

HEMOSTASIS:

Bleeding time= 10 min

INR 0.91 (RI 0.85-1.15)

Howel Gram 2 min

PTT 24.8 sec (RI 23-34)

TT 15.8 sec (RI 13-18)

Chemistry:

BUN 41 mg/dL (RI 9-23)

Creatinine 0.8 mg/dL (RI 0.3-1.0)

Bilirubin Conj. 0.5 mg/dL (RI 0.0-0.3)

Total 2.8 mg/dL (RI 0.0-1.3)

Haptoglobin <5 mg/dL (RI 50-150)

Urinalysis: Large amount of blood present

Protein positive (100 mg/dL)

IMMUNE THROMBOCYTOPENIC PURPURA

Plasma exchange was commenced promptly after admission.

Initially, she became more acutely ill, and developed neurologic symptoms (combative and irritable, with fluctuating levels of consciousness).

She was continued on plasma exchange and given other appropriate therapy.

Over the next several days, her physical and mental status improved,

signs of hemolysis diminished, and her PLT count gradually increased.

She was discharged to be followed in Hematology Clinic.

CASE STUDY 6

History

54 year old female.

One year history of fatigue, weight loss, and

increasingly severe back pain.

Physical Exam

She appeared pale, but otherwise her physical

exam was within normal limits.

PROTEIN ELECTROPHORESIS

Total protein 11.0 g/dL (RI 5.2-8.3)

Serum protein electrophoresis:

Albumin 3.2 g/dL (RI 3.0-5.0)

Globulins:

Alpha1 0.4 (RI 0.1-0.5)

Alpha2 1.0 (RI 0.5-1.2)

Beta 0.8 (RI 0.5-1.1)

Gamma 5.6 (RI 0.6-1.7)

Immunoglobulins, quantitative:

IgA 9 mg/dL (RI 85-450)

IgG 5800 mg/dL (RI 800-1700)

IgM 25 mg/dL (RI 60-370)

Fibrinogen 650 mg/dl

ESR 70 mm/h

MULTIPLE MYELOMA

CASE STUDY 7

History

75 year old male.

Symptoms of severe headache and generalized pruritis.

Physical Exam

Spleen palpable 10 cm. below left costal margin. Liver palpable 3 cm. below right costal margin. The rest of the exam was within normal limits.

PULMONARY FUNCTION:

Oxygen saturation: 97% (RI 94-100)

POLYCYTHEMIA VERA

90

Case study 8

34 year old male. Seen for treatment of superficial skin wounds resulting from a

shotgun accident while grouse hunting. Family physician noted slight pallor, jaundice,

and scleral icterus. History of cholecystectomy five years prior to admission. At that time

the patient was told he had Gilbert's syndrome. He stated he had always had "low

blood," and that his father and paternal grandfather both had "liver ailments."

Physical Exam: Somewhat pale yellowish skin with scattered small surface wounds-

mostly over the face, scalp and upper extremities. Moderate scleral icterus. Spleen

palpable 3 cm below the left costal margin.

CBC (with microscopic differential)

RBC 3.93 x 10[12]/L HGB 11.3 g/dL HCT

33.1 % MCV 84.1 fL

MCH 28.8 pg MCHC 34.4 g/dL

RDW 18.7

WBC 5.0 x 10[9]/L

N 53 % , L 31%, M 8%, E 6%, B 2%

PLT 362 x 10[9]/L

91

Morphologic Alterations

Results of the blood smear exam were:

RBC morphology:

normochromic

2+ polychromasia

2+ anisocytosis

2+ spherocytes

1+ echinocytes

WBC morphology:Within normal limits (one lymphocyte shown

here)

PLT morphology: Within normal limit

Question What further laboratory studies, if any, are indicated?

92

Further Laboratory Studies

Hematology:

Reticulocytes 14.3 % Absolute 562 x 10[6]/L Osmotic fragility (unincubated)

Initial hemolysis 0.65% NaCL

Complete hemolysis 0.40% NaCL Control: Initial 0.50%;

Complete 0.20% Osmotic fragility (incubated) Initial hemolysis 0.85% NaCL

Complete hemolysis 0.60% NaCL Control: Initial 0.60%; Complete 0.20%

Chemistry:

Bilirubin Conj. 0.5 mg/dL (RI 0.0-0.3) Total 5.8 mg/dL (RI 0.0-1.3)

Question What is the most likely diagnosis?

93

Clinical Course

The patient was referred to a hematologist to evaluate the

advisibility of a splenectomy

Answer 3

Diagnosis Hereditary spherocytosis

PRACTICE QUESTIONS

95

Case study 9

37 year old male. Lifelong history of a seizure disorder, treated since age two. At a

routine check with his neurologist, he complained of fatigue, exertional dyspnea, and

lightheadedness over the past 2-3 months. He appeared pale, but otherwise his

physical exam was within normal limits. He was found to have a decreased

hemoglobin, and was referred to Hematology Clinic.

CBC

RBC 1.26 x 10[12]/L

HGB 5.7 g/dL HCT 16.3 %

MCV 130 fL MCH 45.2 pg

MCHC 34.9 g/dL RDW 18.1

WBC 6.2 x 10[9]/L

N 73 % L 21 M 1 E 4 B 1

PLT 219 x 10[9]/L

Question

What morphologic alterations are seen in this blood smear field?

96

Answer 1

RBC morphology:

Normochromic

3+ macrocytosis

3+ anisocytosis

Numerous oval macrocytes

Occ teardrop cells and fragments

WBC morphology: Many neutrophils show nuclear hypersegmentation

PLT morphology: Within normal limits

Question 2

What further laboratory studies, if any, are indicated?

97

Answer 2 Further Laboratory Studies

Bone marrow biopsy Aspirate : Erythroid hyperplasia with megaloblastic maturation.

Large polychromatic and orthochromatic megaloblasts show nuclear karyorrhexis

and other dyserythropoietic changes. Multiple Howell Jolly bodies are seen in both

megaloblasts and oval macrocytes. eutrophils show premature nuclear

segmentation, with giant metamyelocytes and band forms.

Sections:Appear hypercellular

Chemistry:

Serum folate <1.0 µg/L (RI 3.5-15)

RBC folate 131 µg/L (RI 160-600)

Serum B12 136 ng/L (RI 250-900)

Question 3

What is the most likely diagnosis?

98

Clinical Course

The patient was given large doses of folic acid, and within 6 days his reticulocyte

count was 15.2%. One month later, his hemoglobin was 12.7 g/dL, MCV was 92 fL,

and his blood smear morphology was normal. The anticonvulsant drug he had been

taking is known to interfere with folate metabolism. In addition, the patient had

been trying to lose weight, and over the past few months his diet had consisted

mainly of TV dinners, with little or no fresh vegetables or fruits. A nutritional consult

was arranged, and he was instructed to add folic acid to his daily medications.

Note: Patients with folic acid deficiency occasionally show decreased levels of

vitamin B12. Because of the patient's history and lack of typical neurologic

symptoms, concurrent pernicious anemia was considered very unlikely.

Answer 3

Diagnosis Megaloblastic anemia due to folate deficiency

BLOOD

1. is a type of connective tissue

2. is involved in the process of homeothermy

3. bicarbonate buffer system is the most

important extracellular system

4. ADH helps at the reabsorption of Na+ in the

distal nephron

HEMATOCRIT

1. somatic hematocrit is higher than the venous

one

2. represents the % of formed elements in the

blood

3. blood viscosity is inversely proportional to the

hematocrit value

4. splenic venous blood has the highest value

PLASMA PROTEINS

1. albumin is the main contributor to oncotic

pressure

2. the normal concentration of albumin is 7 g/dl

of blood

3. ceruloplasmin is a copper carring protein

4. albumin is a positive acute phase protein

IMMUNOGLOBULINS

1. IgM is released during primary humoral

response

2. IgG may cross the placenta

3. IgA is important in local mucosal immunity

4. IgE trigger alergic reactions

ESR

1. 1. is directly proportional with blood viscosity

2. 2. low ESR can be encountered in anemia

3. 3. it’s measured after centrifuging the blood at

12000 RPM

4. 4. detects non-specific inflammation

RED BLOOD CELLS

1. 1 g of Hb can transport 1.34 mL of oxygen

2. MCV =120fL showes that there are

macrocytes present in the blood

3. 70% of the iron in the organism is found in

hemoglobin

4. when pCO2 is low, red blood cells release O2

more easely to the tissues

WHICH OF THE FOLLOWING ARE PART OF THE

SPECIFIC IMMUNE ANSWERS

1. first line of defense mechanisms

2. second line of defense

3. all three lines of defense

4. third line of defense

CHOOSE THE RIGHT MACROPHAGES FUNCTIONS

1. antigen presenting cells

2. initiation of humoral immune answer

3. initiation of celular immune answer

4. they are the first cells to respond to tissue

infection

ANTIBODIES WORK AS

1. opsonins

2. antitoxins

3. agglutinate bacteria

4. stimulate perforin- pores formation in

antigenic cell membrane

FORMS OF CO2 TRANSPORT IN THE BLOOD ARE

carbaminoHB

bound to albumin

bicarbonates

carboxyHB

CHOOSE THE RIGHT CONDITIONS WHICH SHIFT

THE OXYHB DISSOCIATION CURVE TO THE RIGHT

Hb has low affinity for oxigen

high 2,3 DPG

low pH

low temperature

WHAT WOULD BE THE ERYTHROCYTE INDICES

PROFILE FOR A PERSON WITH NORMOCHROMIC

MACROCYTIC ANEMIA

1. MCHC= 28 g/100 ml

2. MCHC= 35 g/100 ml

3. MCV= 80 fL

4. MCV= 120 fL