Hemostasis – A Beginning

JourneySteve Schmiedel, CLS (ASCP)

Applications Consultant

9/5/2017

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❑ An overview of the coagulation cascade

❑ The importance of balance between thrombotic and

fibrinolytic pathways

❑The most commonly used conventional coagulation tests

❑How a clotting reaction takes place in vitro

Hemostasis 101In this course, you will learn:

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Overview of Hemostasis

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One the systems in the body that

Help to Maintain Normal Blood

Flow

Definition of Hemostasis

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Hemostasis Consists of Coagulation and

Fibrinolysis

Coagulation

means “Forming a Clot”

Fibrinolysis

means “Breaking Down a Clot”

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Thrombotic System

Hemostasis

Hemostasis is the balance of two systems……

Fibrinolytic System

Hemostatic Balance

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Losing the balance

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Activation

Thrombotic System

Hemostasis

The thrombotic system involves the conversion of fibrinogen to fibrin, forming an insoluble clot.

•Preventing blood loss

Thrombotic System

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Activation

Fibrinolytic System

Hemostasis

Fibrinolytic System

The objective of the fibrinolytic system the breakdown of insoluble clots.

•Keeping circulatory system flowing

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Primary Hemostasis’s end result is the process of

forming a platelet plug at the site of vessel injury

Primary Hemostasis

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The initial phase of the process is vascular constriction

Primary Hemostasis

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Next, platelets become activated and aggregate

at the site of injury, forming a temporary, loose

platelet plug, but not sufficient to promote healing.

Primary Hemostasis

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.• A sequence of enzymatic reactions is initiated that

culminates in the formation of fibrin strands

• A fibrin mesh (also called a clot) is formed and

entraps the plug

Secondary Hemostasis

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Tissue Factor ActivatorExtrinsic Pathway

Contact ActivatorIntrinsic Pathway

Thrombin (IIa)

Fibrinogen Fibrin

XII XIIa

XI XIa

IX IXa

Xa

VIIIa + Ca++ + phospholipid

X

VIII

VII

Ca++

VIIa

Va + Ca++ + phospholipid

Prothrombin (II)V

Coagulation Cascade

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The common pathway is activated by either

the extrinsic or intrinsic pathways which

results in the conversion of fibrinogen to fibrin

X Xa

V, PF3, Ca++

(Prothrombin) II IIa (Thrombin)

Fibrinogen Fibrin

Common Pathway

Our Passion.Your Results.

Fibrinolysis is the body’s way of keeping

coagulation from becoming excessive

Fibrinogen FDPs Plasminogen

Tissue

Plasminogen

Soluble Plasmin Activator

Fibrin

XIII

Insoluble (stable)

Fibrin clot D-dimer

(Factor XIII converts soluble Fibrin to Stable Fibrin)

Fibrinolysis

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•For more visual learners, please visit the links below:- https://www.thrombosisadviser.com/the-coagulation-cascade/

- https://www.youtube.com/watch?v=cy3a__OOa2M

17

Additional Resources

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Conventional Coagulation Tests

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Prothrombin Time (PT)

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Prothrombin Time (PT)

•Most commonly performed coagulation assay

•Clot-based assay

•Measures the extrinsic and common pathways- Factors I, II, V, VII and X

•Prolonged when factors fall below 40-50%

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PT for Screening

•Normal; extrinsic thrombotic system functional

•Abnormal (prolonged result)

• Congenital or acquired deficiencies

• FVII, FX, FV, FII or fibrinogen

• Vitamin K (newborns – liver not fully developed with adequate factors)

• Liver disease

• Vitamin K antagonist therapy (e.g. warfarin, Coumadin)

• Inactivates Vitamin K factors II, VII, IX, X

• Others

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Birth of Coumadin – most widely used

anticoagulant

•1920’s – cattle in Canada and Northern American plains

were dying from internal bleeding.

•Grazed on sweet clover hay, but moldy sweet clover hay

was the culprit, via strains of the Penicillin mold.

•“Sweet Clover Disease” was thus discovered - 1941

•Professor Karl-Paul Link at the Wisconsin Alumni

Research Foundation (W.A.R.F) was able to discover that

natural Coumarin in sweet clover became oxyidized by

the mold to become “Dicoumarol” or now called

Coumadin.

•“WARFarin was the early name for Coumadin, and it was

used initially as a rat poison, before being used clinically.23

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Activated Partial Thromboplastin

Time (APTT)

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•2nd most common coagulation test

•Clot-based assay

•Screen for bleeding disorders

-Intrinsic pathway (factors VIII, IX, XI & XII)

- Factors X, V, II, fibrinogen

•Monitor unfractionated heparin (UFH)

therapy

APTT Test

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Historical view of anticoagulant therapy

Heparin•1916 - Discovery of heparin – Dr.

Jay McLean – accidentally while

trying to isolate cephalin.

•1928 – Purified and large scale

production (Dr. Best & team)

•1935 – Human clinical trials started

•1960’s – First randomized trials

published in VTE

Dr. Charles Besthttps://en.wikipedia.org/wiki/

Charles_Herbert_Best

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Wall Street Journal, 2008

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Basis for Anticoagulant Activity

•Heparin binds to antithrombin (AT)

•Converts AT from slow to rapid inhibitor (1000 fold)- Conformational change

•Inactivates thrombin (IIa), Xa, XIIa, XIa and IXa

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Biochemistry of Heparin

•Sulfated glycosaminoglycan (GAG)

-Polysaccharides composed of repeating disaccharide

units

•Heterogeneous-Molecular weight 5,000 – 30,000 daltons

•Derived from porcine or bovine sources

•Because of Mad Cow Disease, porcine sources

are utilized.

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Prolonged APTT

•Most common causes of prolonged APTT -Heparin

-Primary or secondary factor deficiencies (FVIII)

-Specific factor inhibitors

-Non-specific inhibitors

• Lupus anticoagulant or other non-specific antibody

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Activated Clotting Time (ACT)

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Activated Clotting Time (ACT)

•The Activated Clotting Time (ACT) first came into

clinical use in the mid-1970s to guide the

administration and reversal of heparin during

cardiopulmonary bypass procedures

•Measures the time needed for whole blood to clot

upon exposure to an activator (most commonly

Celite, Kaolin, or glass beads)

- This results in the activation of coagulation via the intrinsic [Factor

XII] pathway

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Activated Clotting Time ACT

•Typically used in clinical procedures where the

heparin is required to keep the blood from

clotting during an invasive procedure

•Advantages:

- Faster than APTT

- Can be performed at the bedside

- New automation – Hemochron Elite system - Accriva

•Disadvantages:

- Less precise than the APTT

- Lacks high correlation with the APTT or with heparin anti-Xa levels

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Activated Clotting Time ACT

•Ranges

- The reference range for the ACT varies considerably depending on

the method used for the test; it usually falls somewhere within 70-

180 seconds

- With cardiopulmonary bypass heparinization, the goal is to exceed

400-500 seconds (commonly >480 seconds), depending on the

method, representing a mean heparin level of approximately 4-5

units/mL

- For other indications, the ACT goal is typically lower than it is for

cardiopulmonary bypass