Blood

Chapter Blood  RBC  Platelet  Hemostasis

Section 1

Outline of the Blood

Body fluid

Distribution of the Body Fluid Intracellular fluid (ICF) 40% Interstitial fluid 15% Extracellular fluid Plasma 4% (ECF) 20% Other fluid 1% (lymph, CSF)

Exchange between the capillary blood and the tissue fluid is by formation and reabsorption of tissue fluid and by diffusion into and out of the capillary

Functions of the Blood  Transport 

  

Nutrition and excretion, gases Maintain homeostasis of internal environment Ionic and osmotic balance, buffering Nervous-humoral regulation Protection Hemostasis, anti-infection, immune reaction Heat balance

Blood Volume Blood volume= blood cells + plasma 7% to 8% of body weight. Measurement of blood volume (formula) Blood volume= plasma volume/(1hematocrit) 血浆 / （ 1- 红细胞比容）  The total blood volume an average person is approximately 5.5L

Hematocrit  Hematocrit (the fractions of blood, Packed Cell Volume, PCV) The percentage of the original volume of blood then occupied by the erythrocytes is called the hematocrit ( 比容 )

Hematocrit Male 40%—50%; female 37%—48% Represent relative concentration of RBC Various with the distribution in vessels Decrease in the patients being ill with anemia

Physics and Chemical Properties of Blood 1. pH and Buffers -pH 7.35 ~7.45

-buffer pairs in plasma -buffer pairs in RBC

NaHCO3/H2CO3, Na-prot./Kprot. KHb/HHb KHbO Na 2/HHbO 2 2PO4 2HPO 4/NaH K2HO4/KH2PO4 KHCO3/H2CO3

Physics and Chemical Properties of Blood 2. Viscosity    

（血液的粘度） Viscosity results from friction of liquid internal molecule. water 1
Physics and Chemical Properties of Blood 3. Specific Gravity （血液的比重）

Blood: 1.050-1.060 Plasma: 1.025-1.030 RBC: 1.090-1.092 RBC > Blood > Plasma

Physics and Chemical Properties of Blood 4. Plasma Osmotic Pressure 

Osmotic pressure lies on the number of solut  Plasma osmotic pressure: 300 mmol/L  Plasma osmotic pressure = crystal osmotic pres

Osmosis

Plasma Osmotic Pressure Build up with Role

Normal value

Crystal osmotic pressure inorganic ions ( Na+ , Cl- , K+) & small organic molecules Balance water between inside and outside of the cell

Colloid osmotic pressure protein

285.5mOsm

1.5mOsm

Balance water between inside and outside of the vessel

Plasma Osmotic Pressure  Diffusion of water (solvent) across a selectively permeable membrane, eg, cell membrane.  Important because large volume changes caused by water movement disrupt normal cell function.

Cell shrinkage or swelling ： Isotonic: cell neither shrinks nor swells Hypertonic: cell shrinks (crenation) Hypotonic: cell swells (lysis)

Iso-osmotic Solution & Isotonic Solution Iso-osmotic Solution Isotonic Solution definition have the same have the same osmotic with plasma. osmotic with plasma; RBC can remain normal shape in it. Solute example

Can or can’t go through cell membrane 0.9% NaCl, 1.9% urea

Can go through cell membrane 0.9% NaCl

Section 2 Blood Cell Red Blood Cell Platelets

Red Blood cell (Erythrocyte)  Function of RBC  Properties of RBC  Production of RBC and its regulation

Function of RBC  Transport O2, CO2

 Buffering – KHb/HHb – KHbO2/HHbO2 – K2HO4/KH2PO4 – KHCO3/H2CO3

Properties of RBC 1. Permeability Permeate to O2, CO2, urea, Cl-, HCO32. Reversible deformation ( 可塑性变形）

Volume (90 µ m3): Surface area(140 µ m2) Surface area in ball shaped RBC is 100 µ m2

Properties of RBC

The resistance of RBC to hypotonic solution.

0.9% 0.8%



3. Osmotic Fragility ( 渗透脆性 ) ：

0.46% 0.34%

指红细胞在低渗盐溶液中发生膨胀、破裂和溶血的特性 ，用于表示红细胞对低渗盐溶液的抵抗能力

Properties of RBC 4. Suspension stability ( 红细胞的悬浮稳定性） During a certain time RBC in blood can suspend relatively stably.

Erythrocyte sedimentation rate (ESR, 红 细胞沉降率） : The rate at RBC settle out of suspension in plasma is called ESR.

Measurement of ESR Put anti-coagulated blood in vertical tube, then RBC will sink slowly for its larger density. ESR is expressed by RBC sinking distance during the first hour.  Normal value by Wei’s method: male,0~15mm/h; female,0~20mm/h

Factors that affect ESR in plasma  Rouleaux formation ( 叠连形成）  RBC sticks to each other with concave.  Rouleaux formation increase ESR.  The reason lies in plasma change but not RBC itself.

Factors that affect ESR in plasma  Any plasma change that will affect RBC surface properties will make RBC tend to stick together.  Fibrinogen, some immunoglobulins increase ESR.  Increased ESR is a measure of the acute phase response to a challenge that may be infective, ischemic, traumatic or malignant  ESR increases with age

Production of RBC 

Materials for production of RBC

 Iron( 铁 ): • Old RBC release 95%; Absorption 5% . • The amount of iron absorption is adjusted by intestinal epithelium, depending on the state of the body’s iron balance. • The more iron in the body, the more in the intestinal epithelium, and the less new iron will be absorbed.

Proteins( 蛋白质 ):intake with food Vitamin B12 (cobalamin 钴胺素 ): The absorption of vitamin B12 from gastrointestine requires a protein, intrinsic factor ( 内因子 ) secreted by gastric parietal cell. Be essential for the formation of DAN. Folic acid( 叶酸 ):It is essential for the formation of DAN, and hence for normal cell division since folic acid is required for synthesis of the pyrimidine thymine ( 胸腺嘧啶核苷酸）

Production of RBC The site of RBC production  Bone marrow ( 骨髓 ), specifically the “red” one.  In adulthood it is different from childhood. child: liver, spleen

The Factors That Affect the Production of RBC  Iron intake not enough or lose too much  Vitamin B12 absorption not enough  Intrinsic factor deficiency  Folic acid absorption not enough

Stop DNA synthesis

Regulation of RBC Formation  In adult, there are total 25× 1012 of RBC. The average life span of an erythrocyte is about 120 days.  Almost 1 percent of the body’s erythrocytes are destroyed and must be replaced every day (100 billion cells/day)  A process of negative feedback controlled by erythropoietin (EPO).

Negative Feedback Cycle Stem cells→BFU-E→CFU-E→erythrocyte progenitor cells

EPO

The number of RBC Cells that generate EPO In kidney

↓ O2 deliver to kidneys

↓ O2

High altitudes Chronic lung disease Blood loss (acute or chronic) etc.

Platelets  Overview  Platelet (or thrombocyte): mean diameter 2~4µ m, smallest formed elements in blood  Formed in marrow: 2/3 traveled with blood in vessel, 1/3 stored in liver  Life time 7 to 14 days

The role of platelets  Promote coagulation  Involving hemostasis  Be nutrient to endothelium of capillaries

Properties of platelets  Platelet adhesion

 Platelet aggregation  Platelet release  Platelet contraction (to compact clot mass)

 Platelet absorb coagulation factor

Platelet activation

Platelet adhesion  Platelet can’t adhere to the surface of normal endothelial cells.  Platelet adhesion needs: 1. Glycoprotein (GP, especially GPIb) on platelet membrane; 2. Subendothelium tissue (especially collagen fibers) 3. Von willebrand factor (vWF) in plasma.  collagen fibers—vWF—GPIb

Reagents That Induce Platelets Aggregation  ADP  Thromboxane A2 (TXA2, ，血栓素 A2)  Collagen ( 胶原 )  Thrombin ( 凝血酶）  Pathologic ： bacteria, virus, immune complex, durg, etc.

Reagents That resist Platelets Aggregation

In normal situation, TXA2 and PGI2 keep a dynamic balance.

Platelet release  Platelet can release 5HT, PG, histamin, ATP, PF3, TXA2, etc.  The reason that can induce platelet aggregation can almost also induce its release.  Many of the released substances can promote platelet activation, aggregation and accelerate coagulation.

Section 3 Hemostasis Coagulation Fibrinolysis

Hemostasis

Hemostasis  Definition: the process the body uses to

stop the flow of blood when the vascular system is damaged.  STEPS: 1. Vasoconstriction 2. Platelet plug 3. Blood Coagulation

Hemostasis Is a Two Stage Process • The primary stage is characterized by vascular contraction, platelet adhesion and formation of a soft aggregate plug. • The secondary stage is responsible for stabilizing the soft clot and maintaining vasoconstriction.

Hemostasis Process  Endothelial cell injury and Subendothelium uncover  Platelet activated; Vascular constriction; Coagulation system activated  Platelet plug and fibrin formation

Bleeding time  Definition: Pinpoint pierce into the earlobe or fingertip, then the bleeding lasting time is Bleeding time (BT).  It is a measure about the function of platelet.  Normal value of BT: 1 to 3 min

Coagulation & Fibrinolysis  Blood coagulation (clotting)  Anticoagulant  Fibrinolysis

Blood Coagulation  Definition  a process in which liquid blood is changed into a semisolid mass (a blood clot)

Factor I

Coagulation Factors Plasma halfName life (h)

Fibrinogen

纤维蛋 72 - 96

白原

II

Prothrombin

凝血酶 60

原

III

Tissue Factor or thromboplastin 组织 因子

IV V

Ca++ Proaccelerin

前加速 15

素

VII

Proconvertin 素

前转变 5

Factor

Name

Plasma half-life (h)

IX

Antihemophilic B factor or 25 Christmas factor 血浆凝血激酶

X

Stuart or Stuart-Prower 40 factor Plasma thomboplastin 45-65 antecedent 血浆凝血激酶前质子

XI

XII

Hageman factor, contact factor 接触因子

XIII

Fibrin stabilizing factor 纤维 150 蛋白稳定因子

60

Three essential steps of Clotting  Formation of prothrombinase complex  Prothrombin (FII) activated  Formation of fibrin (Ia) Formation of prothrombinase complex （ FXa-FVa-Ca2+- 磷脂复合物）

II

IIa I

Ia

Formation of prothrombinase complex  Intrinsic Pathway  Extrinsic Pathway  Intrinsic pathway interconnecte with extrinsic pathway

Intrinsic Pathway  Intrinsic Pathway is defined as a cascade that utilizes only factors that are soluble in the plasma.  Surface activation ： a process from

FXII combining with alien substance to FXIa formation.  FXIa activate FIX in the present of Ca2+, then FIXa combine with FVIIIa to form tenase bomples, which can activate FX.

noit a vit ca ecafr u S

Contact with the collagen under subendothelial cell

Extrinsic Pathway  Extrinsic Pathway triggered by tissue

damage, which cause the release of factor III （ Tissue Factor ）  FIII combine with FVII to form FVIIaFIII complex, which activate FX.  Factor III are distributed in various tissue, especially lung, brain, placenta ( 胎盘 ).

noit a vit ca ecafr u S

Contact with the collagen under subendothelial cell

 Intrinsic

pathway interconnect with extrinsic pathway TF-VIIa complex activate FIX.  Amplification of

coagulation FIIa activate FXI.

Formation of fibrin

Summary  The classical model of blood

coagulation involves a series (or "cascade") of zymogen activation reactions.  Coagulation can be initiated via the “intrinsic” or “extrinsic” pathway.

Summary  Both the intrinsic and the extrinsic pathways

proceed through a common pathway by forming activated factor X (factor Xa-VaCa2+)  Amplification of coagulation reactions.  The intrinsic and the extrinsic pathways are highly interconnected.

Intrinsic and the extrinsic pathways interconnect Evidences: 1. The TF-VIIa complex activates not only factor X but also factor IX of the intrinsic pathway.

Factor VIIa activated by TF not only activates factor X but also activates factor IX in the presence of TF, providing a connection between “extrinsic” and “intrinsic” pathway.

Evidences: 2. Patient with severe factor VII deficiency may bleed even though the intrinsic pathway is intact. 3. The severe bleeding associated with deficiencies of factor VIII or IX would not be expected if the extrinsic pathway alone were sufficient to achieve normal hemostasis.

More recent evidence has shown that the pathway are not, in fact, redundant but are highly interconnected.

Factors That Affect Coagulation  Temperature: T ↑ coagulation ↑

 Rough surface  Chemical ： Ca2+ , chelates ( EGTA, EDTA, Sodium citrate, Potassium citrate ）  Biochemical reagents, vitamin K

Anticoagulative System  The reasons that blood can flow through vessels fluently are as follows: – There is no injury in blood vessel. – Most of clotting factors are present in blood with inactive state. – There are some anticoagulants (Compounds that do not allow blood to clot) exist in blood (most important).

Anticoaglants  Endogenous Inhibitors of Clotting 1. serine proteases inhibitor 丝氨酸蛋白酶抑 制物  Antithrombin (major, 抗凝血酶 III): primarily neutralizes factor Xa and thrombin, in addition to inhibiting most active of the clotting system.  α 2 -macroglobulin (α 2- 巨球蛋白）  α 1 -proteinase inhibitor( α 1- 蛋白酶抑 制物）

2. Protein C system  Protein C is another plasma protein that limits clotting by being activated by thrombin to proteolytically inactivate proaccelerin (V) and antihemophilic factor (VIII).  Thrombomodulin ( 凝血酶调制素 ) specifically binds thrombin (II) so as to convert it to a form with decreased ability to catalyze clot formation.

Anticoaglants  Exogenous Inhibitors of Clotting 1. Heparins (肝素 ） 2. Coumarins( 香豆素 ）competitive 3. 1,3-indanedions

inhibitors of vitamin K in the biosynthesis of prothrombin

1. Heparin forms a highaffinity complex with antithrombin (AT-H). 2. The formation of ATH complex greatly increases the rate of inhibition of two principle procoagulant proteases, factor Xa and thrombin (II).

4. Tissue factor pathway inhibitor (TFPI, 组织 因子途径抑制物 , or lipoprotein-associated coagulation inhibitor ）  TFPI in vivo is thought to be synthesized mainly by endothelial cell.  TFPI inhibits the activation of the extrinsic coagulation cascade through its ability to inhibit factor Xa and combine with FVIIaTF complex.

TF

VII

Ca2+

TF—VIIa—TFPI—Xa Complex of inhibitor

Ca2+ (2) Ca2+

TF—VIIa

TF —Xa

IXa

IX

(1)

X

Xa II

Catalysis

TFPI IIa

Convert

Fibrinolysis Breakdown of Blood Clots

Fibrinolysis  Fibrinolysis – Fibrinolysis leads to the breakdown of fibrin clots (blood clots) and is caused by the action of several enzymes – Whenever fibrinolysin is formed in a blood clot, it can cause lysis of clot and also destruction of many clotting factors, thereby causing hypocoagulability of the blood.

Kallikrein ( 激肽释放酶 )

Endothelium

Cl inhibitor

( 内皮细胞）

uPA

Urokinase ( 尿激酶原 )

tPA PAI-1

Plasminogen ( 纤溶酶原）

( 内皮细胞）

α 2-antiplasmin α 2-macroglobulin

Fibrin (or fibrinogen ) Catalysis;

Convert;

Plasmin ( 纤溶酶 ) Fibrin degradation products Inhibit. PAI-1 纤溶酶原 激活物抑制剂

Section 4

ABO Blood Types

The most well known and medically important blood types are in the ABO group.

Discoverer • Karl Landsteiner discovered ABO blood type in 1900 and 1901. • In 1930, he received the Nobel Prize for this discovery. Karl Landsteiner (1868-1943)

ABO blood type  There are four types: A, B, AB, and O decided by the antigens (Antigen A/B) on RBC membrane.  There are two antigens and two antibodies that are mostly responsible for the ABO types.

Antigen and Antibody in ABO Blood Type ABO Blood Type

Antigen Antigen Antibody Antibody A B anti-A anti-B

A

yes

no

no

yes

B

no

yes

yes

no

O

no

no

yes

yes

AB

yes

yes

no

no

Genetic Inheritance Patterns  ABO blood types are inherited through genes on chromosome 9  Do not change as a result of environmental influences during life.  An individual's ABO type is determined by the inheritance of 1 of 3 alleles (A, B, or O) from each parent.

Genotype

AA

AO

BB

BO

AB

OO

Phenotype

A

A

B

B

AB

O

 The alleles must be two of A, B and O to form a person’s Genotype.  Both A and B alleles are dominant over O

Parent Alleles

A

B

O

A

AA (A)

AB (AB)

AO (A)

B

AB (AB)

BB (B)

BO (B)

O

AO (A)

BO (B)

OO (O)

The possible ABO alleles for one parent are in the top row and the alleles of the other are in the left column. Offspring genotypes are shown in white. Phenotypes are red.

Unagglutinated blood smear

Agglutinated blood

Sometimes when the blood of two people is mixed together, it clumps or forms visible islands in the liquid plasma--the red cells become attached to one another.   This is agglutination.

Antibodies seeking specific antigens

Antibodies agglutinating red cells

Anti B

Anti A Anti-A-B (Serum) ABO Blood Type Diagnosis

B

A

AB

O

Blood Transfusion  

Advances: transfusion of blood components Cross-match test

1. Main test: Donor’s RBC + Recipient’s serum 2. Subordination test: Donor’s serum + Recipient’s RBC