1. Blood
Basic Principles of Hemostasis

2. Objectives: Describe the 4 steps of hemostasis
Describe platelets, their normal count, development, and functions.
Describe the process of coagulation
List the natural anti-coagulants present in blood
Define hemophilia and list possible symptoms of hemophilia

3. Steps of Hemostasis Vascular spasm (vasoconstriction)
Hemostasis or haemostasis: is a process which causes bleeding to stop, to keep blood within a damaged blood vessel.
The opposite of hemostasis is hemorrhage which is the first stage of wound healing.
Four steps:
Vascular spasm (vasoconstriction)
Platelet plug formation
Clot formation (blood clotting)
Clot retraction

4. Platelets
Platelets (thrombocytes) are minute discs 1 to 4 μm in diameter.
Formed in the bone marrow from megakaryocytes,
The normal concentration of platelets in the blood is between 150,000 and 400,000 per μL.
COMPONENTS
Actin and myosin molecules.
Residuals of both the Endoplasmic Reticulum and Golgi apparatus.
Mitochondria and enzymes that are capable of forming ATP.
Fibrin-stabilizing factor.
A coat of glycoproteins that repulses adherence to normal endothelium.
Half life of 8 to 12 days.
Factor XIII or fibrin stabilizing factor is an enzyme (EC ) of the blood coagulation system that crosslinks fibrin. Transglutaminase
Cell membrane of platelets is very important in adhesion to the endothelial layer.

5. Platelet or Thrombocyte Physiology
Shape: Biconvex disk like, diameter about 2~4 µm, average cubage 8 µm3.
Complicated structure: under the electronic microscope, there are α-granule, dense body, lysin peroxide enzyme, opening tubular system, dense tubular system, canaliculus,etc.
Dense body: It contains ADP, ATP, 5-HT, Ca2+, epinephrine, etc.
Origin: Platelet comes from megakaryocyte fractionlet release
in the marrow.

6. Functions of platelets
Help in hemostasis (stoppage of bleeding) by:
producing vasoconstriction
2. becoming sticky (activated) to form a platelet plug to close small holes in blood vessels
3. initiate coagulation
Clinical implications:
Low dose Aspirin reduces platelet adhesiveness:
Rationale for use in “blood thinning”
Low platelet count (< 50,000/μL) causes bleeding disorder
Simple investigation: Bleeding time measurement by finger prick.
Normal bleeding time 3-6 minutes
Disease when platelet count is low: Thrombocytopenic purpura

7. Platelet Plug
When platelets come in contact with a damaged vascular surface, they immediately change their own characteristics.
Begin to swell;
Assume irregular forms with numerous irradiating pseudopods;
Release granules that contain multiple active factors;
Become sticky and adhere to collagen in the tissues and to a protein called von Willebrand factor that leaks into the traumatized tissue from the plasma;
Secrete large quantities of ADP;
Enzymes form thromboxane A2.
ADP and thromboxane in turn act on nearby platelets to activate them as well.
von Willebrand factor: vWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue.

8. Under the electronic microscope
Inactive Platelet
Under the electronic microscope

9. Activated Platelet for Hemostasis
Under the electronic microscope

10. Blood coagulation: factors involved

11. Physiological Hemostasis
*Definition: The process from vessel bleeding to automatic hemostasia.
*Bleeding time: The time from vessel bleeding to automatic hemostasia. Normal time is 1~3 min and it is longer when platelet decrease.
* Process of hemostasis:
Blood vessel contraction or convulsion (induced by neuroreflex; 5-hydroxytryptamine (5-HT); thromboxane A2 (TXA2); endothelin (ET ).
Platelet thrombosis forming (made by platelet adhesion, aggregation, release and contraction).
Fibrin, clot forming and maintenance (made by blood coagulation activation).

12. Stable Hemostatic Plug
Hemostasis:
BV Injury
Tissue Factor
Neural
Coagulation
Activation
Blood Vessel
Constriction
Platelet
Activation
Primary hemostatic plug
Reduced
Blood flow
Plt-Fusion
Thromibn,
Fibrin
Stable Hemostatic Plug

13. Physiological Hemostasis

14. Endocrine functions of vessel endothelial cells
Materials related to hemostasis: are basal membrane, collagen (III, IV), microfibril, elastin, laminin, ectonectin, fibronectin, von Willebrand factor (vWF), protein enzyme, protein enzyme inhibitor, adhesive amylose, etc.
Anticoagulative material: They are prostacyclin (PGI2), endothelium-derived relaxing factor (EDRF or nitric oxide, NO), tissue-type plasminogen activator (tPA), uPA, ADPase, ATIII, heparin sulfate, protein C, thrombomomodulin (TM), plasminogen activator (PA).
Promoting coagulative material: Tissue factor, vWF, blood clotting factor V, plasminogen activator inhibitor (PAI-1, PAI-2, ATIII), TNFα, interleukin-1 (IL-1).
Vessel constricting and relaxing modulators: endothelin-1 (ET-1), EDRF (NO), PGI2, etc.

15. Roles of Vessel Endothelial Cells in the Hemostasis
Roles are close related to its endocrine functions
Vessel endothelium serves as barrier between under endothelial structure (namely, collagen) and blood. As soon as collagen expose to blood, hemostasis of platelet is immediately activated to form thrombus blocking wounded vessels.
Platelet activation can releases constrictive factors (TXA2, ET-1, 5-HT, etc) making vessel convulsion, lasting about 60 sec.
Stimulated vessel endothelial cells release coagulative factors and Promoting coagulative material to realize, speed up blood coagulation. At the same time, cells also release anticoagulative factors and fibrinolysis material to modify blood coagulation.

16. Physiological Characteristics of Platelets
Thrombocyte adhesion: its membrane glycoprotein (GP, GPIb/IX and GPIIa/IIIb), collagen (underendothelial structure), vWF (plasma component), fibrinogen are involved in adhesion.
Thrombocyte aggregation: induced by physiological factors such as ADP, thromboxane A2 (TXA2), epinephrine, 5-HT, histamine, collagen, thrombin, prostacyclin,etc and by pathological factors like bacteria, virus, immune complex, drugs, etc.
Thrombocyte release: ADP, ATP, 5-HT, Ca2+ released from dense body, and β-platelet globin, PF4, vWF, fibrinogen, PFV, PDGF, thrombin sensitive protein from α-granule, and acid protein hydrolyzed enzyme, tissue hydrolyzed enzyme from lysosome.
Thrombocyte contraction: Loose platelet thrombus could turn into compact platelet thrombus by Ca2+ release and cytoskeleton movement (filament/canaliculus) within platelet.

17. Roles of Platelet in Hemostasis
Activation of platelet: Stimulus brings about thrombocyte adhesion, aggregation, release and contraction.
Roles of platelet in hemostasis:
Activated platelets supply lecithoid (phospholipid) surface for blood clotting factor and involve in activating factor X and prothrombin.
Surface of platelet membrane combine with many blood clotting factor, such as fibrinogen, FV, FXI, FXIII to speed up coagulation.
Activated platelets release α-granule which contains fibrinogen to intensify fibrin forming and blood coagulation.
Activated platelets contract clot with its contractive protein to solidify blood coagulation.

18. Mechanism(1) of Platelet in Hemostasis

19. Mechanism(2) of Platelet in Hemostasis

20. Blood Coagulation Blood Clotting Factor
Definition: The process of blood flow from flowing liquid to gel or gelatin.
Serum: Light yellow fluid after blood coagulation.
Difference between serum and plasma mainly consists in no fibrinogen in serum.
Blood coagulation: is a series of complicated biochemical reactions with various enzymes.
Blood clotting factor: Material which are directly involved in blood coagulation. There are 12 factors named Roman numerals, except Ca2+, phospholipid，other factors being protein, and except FIII (TF), others are in fresh plasma synthesized by liver with VitK .
Blood clotting enzymes have two type: inactive and activated type (FII, FVII, FIX, FX, FXI, FXII, FXIII).

21. Blood Coagulation
Intrinsic pathway of blood coagulation: All blood clotting factors involved in blood coagulation come from blood. Eyewinker surface with negative charges (collagenin) on the endothelium of blood vessel activates blood F actor XII as beginning of coagulation named surface activation.
Extrinsic pathway of blood coagulation: Stimulus activates tissue factor (FIII) as beginning of coagulation.
Extrinsic pathway of blood coagulation is faster than intrinsic pathway of blood coagulation because its steps are more simple.
*Basic steps of blood coagulation [typical positive feedback]:
Prothrombin activator forming [FXa-Va-Ca2+-phospholipid] Step 1
Prothrombin thrombin Step 2
Fibrinogen fibrin (clot) Step 3
Hemophilia A, B, C in the clinic results from deficiency of FVIII, FIX,
FXI in the blood, respectively.

22. HEMOSTATIC SYSTEM Thrombin Fibrinogen Fibrin INTRINSIC PATHWAY CLOT
EXTRINSIC
PATHWAY
COMMON PATHWAY
Thrombin
Fibrinogen
Fibrin
PLATELETS
CLOT
FIBRINOLYSIS
COLLAGEN
TISSUE FACTOR
VESSEL WALL

23. Mechanism of Blood Coagulation

24. Anticoagulative system in blood
1-Serine protease inhibitors: inhibit the coagulation cascade.
2-Neutralizers of activated coagulation factors (components of protein C system)
1-Serine protease inhibitors:
1-Antithrombin (III).
2-Heparin and heparin like substance.
3-Alpha 1 antitypsin.
4-Alpha 2 macroglobulin
2-Neutralizers of activated coagulation factors :
1-Protein C: synthesized in the liver, vit. K dependant, activated by thrombin.
2-Thrombomodulin.
3-Protein S and C4b-binding

25. Specific inhibitors of clotting factors
1.Antithrombin III
It is a plasma protein that inactivates thrombin by forming an irreversible complex with it.
It resembles alpha 1-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase.
Also, it blocks other serine proteases in the clotting cascade namely, factors XIIa, XIa, IXa, and Xa.

26. 2.Heparin
The inhibitory action of antithrombin III is enhanced by heparin
It is a negatively charged polysaccharide found in mast cells near the walls of blood vessels and on the surfaces of endothelial cells
Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors.
Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors.

27. Electron Micrograph of a Mast Cell
Electron Micrograph of a Mast Cell. Heparin and other molecules in the dense granules are released into the extracellular space when the cell is triggered to secrete.

28. 3. Alpha 1-antitrypsin
which normally inhibits elastase
alpha 1-Antitrypsin activity normally increases markedly after injury to counteract excess elastase arising from stimulated neutrophils.
The mutant a 1-antitrypsin caused the patient's thrombin activity to drop to such a low level that hemorrhage ensued.

29. Fibrinolysis
Definition: is the process wherein a fibrin clot, the product of coagulation, is broken down (clot retraction). Its main enzyme plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver

30. Disorders and Diseases of blood coagulation
1- Hemophilias: are the best-known coagulation factor disorders.
The three main forms are:
Hemophilia C
(factor XI deficiency,
mild bleeding tendency).
Hemophilia A
(factor VIII deficiency)
Hemophilia B
(factor IX deficiency or "Christmas disease")

31. 2. von Willebrand disease
It is the most common hereditary bleeding disorder and is characterized as being inherited autosomal recessive or dominant.
In this disease there is a defect in von Willebrand factor (vWF) which mediates the binding of glycoprotein Ib (GPIb) to collagen.
This binding helps mediate the activation of platelets and formation of primary hemostasis.

32. Some clotting factors; II, IX, VII, X are synthesized in liver
3. Deficiency of Vitamin K
may also contribute to bleeding disorders because clotting factor maturation depends on Vitamin K.
4. Liver diseases:
Some clotting factors; II, IX, VII, X are synthesized in liver
Liver diseases deficiency of these factors bleeding disorders.