Vasoconstriction, the platelet cell membrane, the formation of a platelet plug, and the significance of the platelet mechanism for sealing vascular holes. PHARMACOLOGICAL AGENTS, INTERACTION BETWEEN THE INTRINSIC AND EXTRINSIC PATHWAYS, BLOOD CLOT, AND THE MECHANISM OF BLOOD COAGULATION

1. HEMOSTASIS
PRESENTATION BY
S.R. BHARATHKUMAAR,
I M.SC BIOTECHNOLOGY 2019-2021 ,
BHARATHIAR UNIVERSITY.

3. Hemostasis:
 The stoppage of bleeding or hemorrhage. Also, the stoppage of
blood flow through a blood vessel or organ of the body.
 Hemostasis is the arrest of bleeding, whether it be by normal
vasoconstriction (the vessel walls closing temporarily), by an
abnormal obstruction (such as a plaque) or by coagulation or
surgical means (such as ligation).
 The term comes from the Greek roots heme, blood + stasis, halt =
halt of the blood.

4. Events in Hemostasis
Hemostasis means prevention of blood loss, Hemostasis achieved several mechanism
1) Vasoconstriction
2) Formation of platelet plug
3) Blood coagulation, or formation of a fibrin clot
4) Growth of fibrous tissue in the clot

5. Vasoconstriction
• Vasoconstriction is produced by vascular smooth muscle cells, and is the blood
vessel's first response to injury.
• The smooth muscle cells are controlled by vascular endothelium, which
releases intravascular signals to control the contracting properties.
• When a blood vessel is damaged, there is an immediate reflex, initiated by local
sympathetic pain receptors, which helps promote vasoconstriction.
• The damaged vessels will constrict (vasoconstrict)
which reduces the amount of blood
flow through the area and limits
the amount of blood loss.

6. • Collagen is exposed at the site of injury, the collagen promotes platelets to adhere to
the injury site.
• Platelets release cytoplasmic granules which contain Serotonin, ADP and thromboxane
A2, all of which increase the effect of vasoconstriction.
• The spasm response becomes more effective as the amount of damage is increased.
• Vascular spasm is much more effective in smaller blood vessels.

7. Development : from the pluripotentstem cell in the bone marrow
CFU-M colony forming megakaryocytes

9. NORMAL CONCENTRATION:
1,50,000 to 3,00,000 per microliter.
Structure
Cell membrane
Cytoplasm

10. CELL MEMBRANE OF PLATELET
 It is 6nm thick and contains lipids (phosholipids,
cholestrol and glycolipids), Carbohydrates (glyocalyx),
proteins and glycoproteins
 Out of all glycoprotein and phospholipids are
functionally important

11. Glycoproteins
 Prevents the adherence of platelets to normal
endothelium
 Accelerates the adherence of platelets to collagen and
damaged endothelium in rupture blood vessels
 Forms a receptor for ADP and thrombin

12. CYTOPLASM
The cytoplasm of the platelet include:
• Golgi apparatus
• ER
• Mitochondria
• Microvessels
• Microtubule
• Microfilaments
• Granules
Cytoplasm also contains
• Proteins
• Enzymes
• Hormones
• Chemical substances

13. PROTEINS
The major proteins are contractile proteins which are responsible
for the contraction of platelets:
• Actin
• Myosin
• Thrombosthenin
CHEMICAL SUBSTANCES
• Calcium ions
• Mg- ions
• Adenosine triphosphate (ATP)
• Adenosine diphosphate (ADP)

14. FUNCTION OF PLATELETS
 Its surface has glycoprotein coat that adhere it to injured
endothelial cells.. Preventing bleeding
 Actin, myosin& thrombostheninn that are contractile proteins..
Cause clot retraction
 Secretes growth factor that promotes growth&multiplication of
vascular endothelial cells, vascular smooth cells& fibrblasts…
repair damaged vascular wall
 Its membrane has phopholipids that activate intrinsic system of
blood clotting

15. LIFE SPAN OF PLATELETS
Platelets are eliminated from the circulation mainly by
the tissue macrophage system in the spleen. Its half time
is 8 to 12 days

16. Formation Platelet Plug
Platelet Adhesion
Platelet Activation
Platelet Aggregation

17. PLATELET ADHESION
-{vWF} von willebrand factor – immobilizes under endothelial damage
-Collagen (hemodynamic changes)
• They have AI in its structure ; while platelet have GPIb-IX-V complex
receptor this complex will got GPIb alpha I subunit and this subunit can
bind with AI subunit of vWF domain
• In the collagen platelet interaction collagen displays alpha 1 CB3 peptide
• Platelet which have a cell surface receptor of alpha 2 beta 1 integral
• During endothelial damage alpha 2 beta integral 1 receives alpha 1 CB3
peptide of collagen and mediate them, that platelet enhnance the damaging
site
• And also GPVI proteins
interacts with collagen

18. PLATELET ACTIVATION
• Glycoprotein VI or GP-6; it is receptor for collagen , it mediates
collagen induced released of several factors
• ADP releases from dense granules, vWF from alpha granules
and also secretion of thromboxane -2
• ADP mediates in two pathways
Purinogenic signalling (receptors)
• P2Y12 receptor and P2Y1 receptor
• P2Y12 is belongs to Gi- Class of GPCR ; P2Y1 is belongs of
Gq class of GPCR

19. Gs Alpha subunit: cAMP dependent pathway
Gi Alpha subunit: inhibits cAMP pathway
Gq Alpha subunit: IP3 pathway
G12 Alpha subunit: Rho family GTPase signalling

20. • ADP molecules binds the P2Y12 receptor , it cause activation of
GPCR by GDP to GTP
• Gi have a cAMP synthesis
• Activation of alpha subunit, inhibit the adenyl cylase which
inhibit the cAMP pathway
• cAMP being the seconary messenger which enter and
inactivates
Which Accumulate the calcium inside platelets off inactivating
• Calcium effective pump platelet activation

21. When ADP binds with P2Y1 it cause activation of Gq of GPCR
by GDP to GTP
PLC activation and it activates….
IP3 pathway
Intracellular release of calcium also cause platelet activation
PLC activation also have drives the granule secretion from
platelet
Its seecretion includes serotonin/PDGF, TXA-2, ADP/PAF
Serotonin and TXA-2 is mainly for vasoconstriction
TXA-2 and ADP is for platelet activation

22. PLATELET AGGREGATION
Platelet aggregation by ADY; which leads to Aformentioned confirmational
changes in platelet receptors
GPII b / III a receptors on platelets
It’s a conformational change upon platelet activation
TXA-2 secreted by activated platelet
Stimulates other platelets via, paracrinesignalling which lead to platelet aggregation
GPII b / III a receptors binds fibrinogen
Platelets stick together
Leads to platelet aggregation

24. Importance of the Platelet Mechanism for Closing Vascular Holes.
 This mechanism is extremely important for closing minute ruptures in very small
blood vessels that occur many thousands of times daily.
 Indeed, multiple small holes through the endothelial cells themselves are often
closed by platelets actually fusing with the endothelial cells to form additional
endothelial cell membrane.
 Literally thousands of small hemorrhagic areas develop each day under the skin
and throughout the internal tissues of a person who has few blood platelets.
 This phenomenon does not occur in persons with normal numbers of platelets.

25. BLOOD COAGULATION IN THE RUPTURED VESSEL
 The clot begins to develop in 15 to 20 seconds -been minor
 1 to 2 minutes –severe
 Activator substances from platelets, and from blood proteins
adhering to the traumatized vascular wall initiate the clotting
process.
 Platelets also play an important role in this clot retraction

27. MECHANISM OF BLOOD COAGULATION
GENERAL MECHANISM
Clotting takes place in three essential steps:
1. In response to rupture of the vessel or damage to the blood itself, a complex
cascade of chemical reactions occurs in the blood involving more than a
dozen blood coagulation factors. The net result is formation of a complex of
activated substances collectively called prothrombin activator.
2. The prothrombin activator catalyzes conversion of prothrombin into
thrombin.
3. The thrombin acts as an enzyme to convert fibrinogen into fibrin fibers that
enmesh platelets, blood cells, and plasma to form the clot.

28. CONVERSION OF PROTHROMBIN TO THROMBIN
 First, prothrombin activator is formed as a result of rupture of a blood vessel or as a
result of damage to special substances in the blood.
 Second, the prothrombin activator, in the presence of sufficient amounts of ionic calcium
(Ca++), causes conversion of prothrombin to thrombin
 Third, the thrombin causes polymerization of fibrinogen molecules into fibrin fibers
within another 10 to 15 seconds.
 Thus, the rate-limiting factor in causing blood coagulation is usually the formation of
prothrombin activator and not the subsequent reactions beyond that point, because these
terminal steps normally occur rapidly to form the clot.
 Platelets also play an important role in the conversion of prothrombin to thrombin
because much of the prothrombin first attaches to prothrombin receptors on the
platelets already bound to the damaged tissue.

29. Prothrombin and Thrombin.
 Prothrombin is a plasma protein, an α2-globulin, having a molecular weight of 68,700.
 It is present in normal plasma in a concentration of about 15 mg/dl.
 It is an unstable protein that can split easily into smaller compounds, one of which is
thrombin, which has a molecular weight of 33,700, almost exactly one half that of
prothrombin.
 Prothrombin is formed continually by the liver, and it is continually being used
throughout the body for blood clotting.
 If the liver fails to produce prothrombin, in a day or so prothrombin concentration in
the plasma falls too low to provide normal blood coagulation.
 Vitamin K is required by the liver for normal activation of prothrombin, as well as a
few other clotting factors.
 Either lack of vitamin K or the presence of liver disease that prevents normal
prothrombin formation can decrease the prothrombin to such a low level that a
bleeding tendency results.

30. CONVERSION OF FIBRINOGEN TO FIBRIN—FORMATION OF THE CLOT
 Fibrinogen Formed in the Liver Is Essential for Clot Formation.
Action of Thrombin on Fibrinogen to Form Fibrin.
 Thrombin is a protein enzyme with weak proteolytic capabilities.
 It acts on fibrinogen to remove four low-molecular-weight peptides from each molecule
of fibrinogen, forming one molecule of fibrin monomer that has the automatic capability
to polymerize with other fibrin monomer molecules to form fibrin fibers.
 Therefore, many fibrin monomer molecules polymerize within seconds into long fibrin
fibers that constitute the reticulum of the blood clot.
 The fibrin monomer molecules are held together by weak non-covalent hydrogen
bonding, and the newly forming fibers are not cross-linked with one another
 Therefore, the resultant clot is weak and can be broken apart with ease.
 However, another process occurs during the next few minutes that greatly strengthens
the fibrin reticulum. This process involves a substance called fibrin-stabilizing factor

31. BLOOD CLOT
 The clot is a meshwork
 Fibrin fibers also adherse to damaged surfaces of blood vessels
CLOT RETRACTION- SERUM
 Contraction causes expression of fluid from clot-serum
 Platelet contractile proteins contributes greatly to the clot
retraction by activting Platelet Thrombosthenin
 They also compress fibrin meshwork into smaller mass
 As the clot contracts, the edges are further pulled together,
contributing ultimate state of Hemostasis

32. INITIATION OF COAGULATION
FORMTION OF PROTHROMBIN ACTIVATOR
Three mechanism are set into play by
Trauma to the vascular wall and the adjacent tissues
Contract of the blood with damaged endothelial cells
They formed in two ways
• Extrinsic pathway for initiating blood clotting
• Intrinsic pathway for initiating blood clotting

35. N

36. ROLE OF CALCIUM IONS IN BOTH PATHWAYS
• Except for the first steps in the intrinsic pathway Ca2+
ions for the promotion/ acceleration of all the blood-
clotting reactions
• In the living body Ca2+ ion concenrtion seldom falls
low enough to significantly affect the kinetics

37. INTERACTION B/W THE INTRINSIC AND EXTRINSIC
PATHWAYS
- SUMMARY OF INITIATION
LYSIS OF BLOOD CLOTS
Plasminogen or profibrinolysin when activated forms
plasmin(fibrinolysin)
Plasmin is a proteolytic enzyme
that digests fibrin fibers protein
coagulants such as fibrinogen,
Factor V, Factor VIII, prothrobin
and Factor XII

38. MECHANISM OF HEMOSTASIS
Mechanical
Chemical
Thermal

39. MECANICAL HEMOSTASIS
 Direct pressure
 Gauze pack
 Suture and ligation
 staples

40. THERMAL ENERGY METHOD
• Heat (cautery)
• Electo cautery: it is the use of high frequency alternating current for
cutting, coagulation, dissication or fulgurating tissue in both open
and laproscopic procedure
• Monopolar electro surgery
• Bipolar electro surgery
• Bipolar electro surgery vessel sealing technology
• Argon enhanced coagulation technology
• Ultrasonic device
• Lasers

41. CHEMICAL METHODS
 Pharmacological agents
 Topical hemostatic agent
 Passive
 Active

42. PHARMACOLOGICAL AGENTS
• Sterile haemocoagulase solution
• Epinephrine
• Vitamin K
• Protamine
• Desopressin
• Lysin analogs
• Etamsylate

43. TOPICAL HEMOSTATIC AGENTS
Passive - collagen based product
- oxidised regenerated cellulose
- gelatine
Active
- thrombin product
- pooled human plasma thrombin
- recombinant thrombin

45. Thank you…..