BIOCHEMISTRY & CELL BIOLOGY

SELF-INSTRUCTION MODULE 3-5

VITAMIN K & HEMOSTASIS

BY: DR. RICHARD HANN

DEPARTMENT OF BIOCHEMISTRY
2013 by Universidad Central del Caribe
All rights reserved
PURPOSE & SCOPE OF THE SELF-INSTRUCTION MODULES

Due to the limitation of faculty-student contact hours, it is not

possible to present in the classroom all of the concepts in
Biochemistry & Cell Biology that the student must learn. The
self-instruction modules (SIMs) are designed to provide,
within a student-centered electronic format, a structured
presentation of essential concepts & information that cannot
be presented in the classroom. Each SIM is a power-point
presentation that the student may access via the Blackboard
BCB course site at his or her convenience. Studying each of
the SIMs is compulsory. The material on each SIM is tested on
the subsequent examinations.
 CONTENTS OF THIS MODULE:

1. INTRODUCTION
2. FORMATION OF A FIBRIN CLOT
3. ROLE OF VITAMIN K IN CLOT FORMATION

4. VITAMIN K DEFICIENCY &

ORAL ANTICOAGULANT THERAPY

5. THE HEMOPHILIAS
 LEARNING OBJECTIVES OF THIS MODULE
Upon completion of this module the student should be able to:

1. Outline in detail how the plasma coagulation factors

lead to the formation of a fibrin clot

2. Explain the role of vitamin K in the synthesis of the

vitamin K-dependent blood coagulation factors &
their function in hemostasis

3. Discuss important nutritional aspects of vitamin K

& the major effects of vitamin K deficiency

4. Describe the molecular causes of the diseases

known as the hemophilias
 1. INTRODUCTION
BLOOD COAGULATION OR CLOTTING RESULTS
FROM THE FORMATION OF A CLOT OR THROMBUS

THE MAIN COMPONENTS OF A BLOOD CLOT ARE A

CELLULAR COMPONENT (PLATELETS) & A HUMORAL
OR PLASMA PROTEIN COMPONENT (FIBRIN)

THE PLASMA PROTEINS THAT PARTICIPATE IN THE PROCESS

OF FORMING THE INSOLUBLE PROTEIN FIBRIN ARE CALLED
THE COAGULATION FACTORS OR CLOTTING FACTORS
 2. FORMATION OF A FIBRIN CLOT

THE INSOLUBLE PROTEIN FIBRIN IS MADE

FROM THE SOLUBLE PLASMA PROTEIN
FIBRINOGEN

SHORT PEPTIDES ARE TRIMMED FROM

FIBRINOGEN BY THE PROTEOLYTIC
ENZYME THROMBIN

FIBRIN MONOMERS SPONTANEOUSLY

AGGREGATE TO FORM A
SOLUBLE OR SOFT CLOT

FIBRIN MONOMERS ARE COVALENTLY

CROSS-LINKED TO FORM A HARD CLOT
cross-linking
(THIS MECHANISM RESEMBLES
Fibrin Hard Clot COLLAGEN FIBRIL FORMATION)
THE FORMATION OF FIBRIN INVOLVES A CASCADE OF PLASMA
PROTEINS CALLED THE COAGULATION FACTORS
EACH FACTOR IS ASSIGNED A ROMAN NUMERAL AS WELL AS A NAME
THERE ARE 11 PROTEIN COAGULATION FACTORS NUMBERED I - XIII
ACTIVE FACTORS ARE INDICATED WITH a
FACTOR I IS FIBRINOGEN
FACTOR Ia IS ACTIVATED FIBRIN MONOMER
THAT FORMS THE SOFT CLOT

FACTOR IIa
IS THROMBIN

FACTOR XIII IS FIBRINOLIGASE THAT

COVALENTLY CROSSLINKS THE FIBRIN
MONOMERS INTO A HARD CLOT
FACTOR XIIIa FORMS AMIDE BONDS BETWEEN
GLUTAMINE & LYSINE ON THE FIBRIN MONOMERS
 THROMBIN CIRCULATES IN THE BLOOD AS AN INACTIVE
PROENZYME OR ZYMOGEN CALLED PROTHROMBIN (FACTOR II)

PROTHROMBIN IS PROTELYTICALLY
ACTIVATED TO THROMBIN (IIa) BY
FACTOR X (THE STUART FACTOR)
THIS REQUIRES THE FORMATION OF A
COMPLEX THAT IS ATTACHED VIA
Ca++ IONS TO A NEGATIVELY-CHARGED
PHOSPHOLIPID SURFACE (PL)
A COFACTOR PROTEIN (FACTOR V OR
PROACCELERIN) IS ALSO NEEDED
THIS SURFACE COMPLEX IS
ESSENTIAL FOR THIS REACTION TO
OCCUR AT A PHYSIOLOGICALLY
SURFACE ATTACHMENT ACCELERATES RELEVANT RATE
THE REACTION BY ABOUT 106 TIMES !
 NOTICE THAT IN THE LIST OF
COAGULATION FACTORS THERE IS NO
FACTOR IV OR FACTOR VI
IN THE ORIGINAL LIST OF
COAGULATION FACTORS THERE WAS A
FACTOR IV & A FACTOR VI

FACTOR IV WAS LATER FOUND TO BE

Ca++ IONS & FACTOR VI CAN BE ANY
NEGATIVELY-CHARGED
PHOSPHOLIPID SURFACE (PL)

SO THESE 2 NUMERALS ARE

NO LONGER USED

FACTOR IV FACTOR VI
FACTOR X CAN BE ACTIVATED EITHER BY FACTOR VIIa OR BY FACTOR IXa
 FACTOR VII (PROCONVERTIN) IS ACTIVATED WHEN
THE BLOOD CONTACTS EXTRAVASCULAR TISSUE
FACTOR VII IS CONFORMATIONALLY ACTIVATED BY
CONTACT WITH TISSUE FACTOR (FACTOR III)
FACTOR VIIa THEN ACTIVATES FACTOR X
THIS IS KNOWN AS THE EXTRINSIC PATHWAY OR EXTRINSIC SYSTEM

TISSUE FACTOR IS AN
EXTRAVASCULAR
PROTEIN THAT NON-
PROTEOLYTICALLY
ACTIVATES FACTOR VII

AGAIN Ca++ & AN ANIONIC PL SURFACE ARE ESSENTIAL FOR THIS

REACTION TO OCCUR AT A PHYSIOLOGICALLY-RELEVANT RATE
 FACTOR IX (CHRISTMAS FACTOR) IS ACTIVATED BY FACTOR XIa
FACTOR IXa FORMS A COMPLEX WITH FACTOR X & FACTOR VIII (THE
ANTIHEMOPHILIC FACTOR) ATTACHED VIA Ca++ TO A PL SURFACE

FACTOR IXa THEN PROTEOLYTICALLY

ACTIVATES FACTOR X

FACTOR XI (PLASMA THROMBOPLASTIN

ANTECEDENT) IS PROTEOLYTICALLY
ACTIVATED BY FACTOR XII (THE
HAGEMAN FACTOR)
FACTOR XII IS ACTIVATED WHEN BLOOD
FLOW SLOWS & ALLOWS XII TO
CONTACT THE VESSEL SURFACE

THIS IS KNOWN AS THE INTRINSIC

PATHWAY OR INTRINSIC SYSTEM

FACTOR VII CAN ALSO

ACTIVATE FACTOR IX
 3. ROLE OF VITAMIN K IN
FIBRIN CLOT FORMATION
VITAMIN K IS REQUIRED FOR THE BIOSYNTHESIS
OF 4 COAGULATION FACTORS

FACTORS II, VII, IX & X ARE THE

VITAMIN K-DEPENDENT ZYMOGENS

FACTORS XI, XII & XIII ALSO

CIRCULATE AS ZYMOGENS
BUT THEY ARE NOT
VITAMIN K-DEPENDENT

FACTORS III, V & VIII ARE

COFACTOR PROTEINS WITH NO
ENZYME ACTIVITY & ARE NOT
VITAMIN K-DEPENDENT
PROTEOLYTIC ACTIVATION OF ALL 4 VITAMIN K-DEPENDENT ZYMOGENS
REQUIRES THE FORMATION OF A COMPLEX THAT IS ATTACHED TO A
NEGATIVELY-CHARGED PHOSPHOLIPID SURFACE
THE PROTEINS ARE HELD ONTO
THE SURFACE BY Ca++ IONS
TO BIND Ca++ WELL THE
PROTEINS MUST HAVE STRONGLY
NEGATIVE AMINO ACID RESIDUES

ON THESE PROTEINS SPECIFIC
GLUTAMATE (GLU) RESIDUES ARE
CHANGED TO
-CARBOXYGLUTAMATE (GLA)

COO OOC COO
EACH -CARBOXYGLUTAMATE RESIDUE
HAS 2 NEGATIVE CHARGES INSTEAD OF 1
GLU GLA
 PRECURSORS OF THE VITAMIN K-DEPENDENT
FACTORS II, VII, IX & X ARE SYNTHESIZED IN THE LIVER
PRECURSORS* ZYMOGENS
WEAK Ca++ BINDING STRONG Ca++ BINDING
II* VITAMIN K II
HEPATOCYTE X* X SECRETION PROTEOLYTIC
SYNTHESIS VII* GLUTAMATE VII TO BLOOD ACTIVATION
IX* -CARBOXYLATION IX
POST-TRANSLATIONAL GLUTAMATE -CARBOXYLATION OF THE
PRECURSORS OF FACTORS II, VII, IX & X IS ALSO DONE IN THE LIVER

VITAMIN K IS THE COENZYME FOR GLUTAMATE -CARBOXYLATION

THE ZYMOGENS WITH -CARBOXYGLUTAMATE RESIDUES ARE SECRETED

INTO THE BLOOD TO BE PROTEOLYTICALLY ACTIVATED AS NEEDED
THE QUINOL OR REDUCED FORM OF VITAMIN K (KH2) IS THE ACTIVE FORM
THAT SERVES AS THE COENZYME FOR THE -CARBOXYLATION REACTION
COO
OOC COO

GLU GLA

KH2 KO

THIS IS AN OXIDATIVE CARBOXYLATION REQUIRING O2 & HCO3

THE ENZYME IS A CARBOXYLASE-EPOXIDASE BECAUSE IT CARBOXYLATES
GLUTAMATE & FORMS THE EPOXIDE OF VITAMIN K (KO)
 KO-REDUCTASE CONVERTS THE EPOXIDE TO THE QUINONE FORM

THE QUINONE FORM OF

VITAMIN K IS ALSO THE
MAIN FORM IN THE DIET
& MADE BY OUR
KH2 KO
INTESTINAL BACTERIA
ACTIVE DIET
FORM
INTESTINAL
BACTERIA

K-REDUCTASE CONVERTS THE QUINONE

TO THE QUINOL OR ACTIVE FORM (KH2)
 4. VITAMIN K DEFICIENCY &
ORAL ANTICOAGULANT THERAPY
CLINICAL CORRELATION
VITAMIN K DEFICIENCY

MAIN CLINICAL MANIFESTATIONS:

EASY BRUISABLITY

BRUISES, HEMATOMAS & ECCHYMOSES

BLEEDING & HEMORRHAGE ECCHYMOSES ON LEFT ARM OF

VITAMIN K DEFICIENT PATIENT
PROLONGED CLOTTING TIME

PROLONGED PROTHROMBIN TIME (PT)

PROLONGED PARTIAL THROMBOPLASTIN TIME (PTT)

 IN VITAMIN K DEFICIENCY LIVER GLUTAMATE -CARBOXYLATION OF
THE PRECURSORS OF FACTORS II, VII, IX & X IS REDUCED

PRECURSORS* ZYMOGENS
WEAK Ca++ BINDING STRONG Ca++ BINDING
II* K II
HEPATOCYTE
SYNTHESIS
X*
VII* GLUTAMATE
X
VII
IX* -CARBOXYLATION IX
SECRETION
TO BLOOD X
PROTEOLYTIC
ACTIVATION

AS A RESULT THE BLOOD LEVELS OF FACTORS II, X, VII & IX

ARE GREATLY REDUCED

THE TIME NEEDED FOR THE BLOOD TO

CLOT IS GREATLY PROLONGED

THIS CAN LEAD TO BLEEDING & HEMORRHAGE

 CLINICAL CORRELATION
HEMORRHAGIC DISEASE OF THE NEWBORN

NEWBORNS ARE ESPECIALLY AT RISK FOR VITAMIN K DEFICIENCY

INADEQUATE ORAL FEEDING OFTEN LIMITS THEIR DIETARY INTAKE

INTESTINAL BACTERIA NORMALLY PROVIDE A SUBSTANTIAL AMOUNT OF

THE DAILY NEED FOR VITAMIN K BUT

A NEWBORNS INTESTINE IS STERILE AT BIRTH

IT TAKES SEVERAL DAYS TO ESTABLISH THE NORMAL INTESTINAL FLORA

EXTENDED ANTIBIOTIC THERAPY IN ANEWBORN PREVENTS GROWTH OF

THE NORMAL INTESTINAL FLORA THAT PROVIDE VITAMIN K
 THE RISK OF NEWBORN VITAMIN K DEFICIENCY IS INCREASED
IF THE MOTHER DOES NOT RECEIVE ADEQUATE VITAMIN K

THIS IS WHY THE FOOD & NUTRITION COUNCIL OF

THE NATIONAL ACADEMY OF SCIENCES RECOMMENDS THAT:

1) ALL PREGNANT MOTHERS RECEIVE VITAMIN K BY INJECTION

DURING THE LAST MONTH OF THE PREGNANCY
2) ALL NEWBORN INFANTS SHOULD GET VITAMIN K BY INJECTION AT BIRTH
BEFORE THESE RECOMMENDATIONS WERE ISSUED
THE INCIDENCE OF VITAMIN K DEFICIENCY IN NEWBORNS IN THE U.S.
WAS ABOUT 1 PER 400 LIVE BIRTHS

THIS INFANT DID NOT RECEIVE

VITAMIN K AT BIRTH BECAUSE
THE PARENTS REFUSED THE
TREATMENT. NOTE THE LARGE
HEMATOMA ON THE LEFT SCALP.
SCALP SUTURES ARE FROM
SURGICAL DRAINAGE OF A
SUBDURAL HEMATOMA WHICH
LEFT RESIDUAL BRAIN DAMAGE.
 CLINICAL CORRELATION
ORAL ANTICOAGULANT THERAPY
THE MOST COMMONLY USED ORAL ANTICOAGULANT
IS WARFARIN OR COUMADIN

COUMADIN IS A COMPETITIVE
INHIBITOR OF K-REDUCTASE

KH2 KO COUMADIN PRODUCES A

DEFICIENCY OF THE ACTIVE
ACTIVE DIET
FORM FORM OF VITAMIN K
INTESTINAL
BACTERIA THIS REDUCES THE LIVER
-CARBOXYLATION OF FACTORS
II, VII, IX & X PRECURSORS
AS IN VITAMIN K DEFICIENCY
COUMADIN
(WARFARIN) THIS REDUCES THE PLASMA
LEVELS OF FACTORS II, VII, IX & X
& SLOWS COAGULATION
 COUMADIN IS ONE OF THE MOST WIDELY USED MEDICATIONS
THE RISK OF COUMADIN OVERDOSAGE IS HIGH
PATIENTS ON COUMADIN MUST HAVE THEIR
CLOTTING TIME CHECKED FREQUENTLY

WARFARIN IS ALSO WIDELY

USED AS A RODENT POISON

KH2 KO WARFARIN IS INGESTED BY THE

RODENT & IT BLEEDS TO DEATH
ACTIVE DIET
FORM
I

INTESTINAL
WARFARIN IS ONE OF THE MOST
BACTERIA
COMMON POISONS IN THE HOME

WARFARIN IS A FREQUENT
CAUSE OF ACCIDENTAL
COUMADIN POISONING & HEMORRHAGIC
(WARFARIN)
DEATH IN CHILDREN
 5. THE HEMOPHILIAS
CLINICAL CORRELATION

HEMOPHILIA A OR CLASSIC HEMOPHILIA

IS CAUSED BY DEFICIENCY OF FACTOR VIII
(THE ANTIHEMOPHILIA FACTOR)

HEMOPHILIA B OR CHRISTMAS DISEASE IS

CAUSED BY DEFICIENCY OF FACTOR IX
(THE CHRISTMAS FACTOR)

FACTOR IX IS A VITAMIN K-DEPENDENT ZYMOGEN

FACTOR IXa ACTIVATES FACTOR X

FACTOR VIII IS A COFACTOR PROTEIN THAT IS ESSENTIAL

FOR FACTOR IXa TO ACTIVATE FACTOR X

FACTOR IX WAS DISCOVERED IN A YOUNG ENGLISH PATIENT

NAMED STEVEN CHRISTMAS WHO WAS A BLEEDER
STEVEN WAS THOUGHT TO HAVE CLASSIC HEMOPHILIA BUT HE DID NOT
 HEMOPHILIA IS A DISEASE THAT CHANGED HISTORY

BOTH THE FACTOR VIII GENE & THE FACTOR IX GENE

ARE ON THE X CHROMOSOME

HEMOPHILIA INHERITANCE IS X-LINKED RECESSIVE

QUEEN VICTORIA WAS A CARRIER OF HEMOPHILIA

HER YOUNGEST SON PRINCE LEOPOLD WAS A

BLEEDER & DIED FROM HEMOPHILIA

VICTORIA QUEEN OF HER OLDEST SON PRINCE EDWARD INHERITED

GREAT BRITAIN THE GOOD GENE FROM HIS MUM & BECAME
1837 - 1901 KING EDWARD VII

PRINCE
LEOPOLD

KING
EDWARD
VII
 ALEXANDRA VICTORIAS GRANDAUGHTER PRINCESS
ALEXANDRA WAS ALSO A CARRIER
ALEXEI
ALEXANDRA MARRIED NICHOLAS II
THE LAST CZAR OF RUSSIA
THEIR ONLY SON ALEXEI HAD HEMOPHILIA
NICHOLAS
ALEXEI WAS HEIR TO THE RUSSIAN CROWN

EFFORTS TO HIDE THAT ALEXEI WAS A BLEEDER ALIENATED THE ROYAL

FAMILY FROM THE RUSSIAN PEOPLE AND CONTRIBUTED TO THE 1917
RUSSIAN REVOLUTION & THE ABDICATION OF NICHOLAS II

ALEXEI NEVER BECAME CZARBUT HE DID NOT DIE FROM HEMOPHILIA

THE ENTIRE RUSSIAN ROYAL FAMLY WAS

ASSASSINATED BY THE BOLSHEVIKS IN 1918
THEIR REMAINS WERE SECRETELY BURIED
IN AN UNDISCLOSED LOCATION
IT HAS ALWAYS BEEN ASSUMED THAT THE ROYAL
DISEASE WAS THE MORE COMMON CLASSIC
HEMOPHILIA OR FACTOR VIII DEFICIENCY

BUT IN 2009 A RUSSIAN GROUP REPORTED ON

MOLECULAR GENETIC ANALYSIS OF DNA
ISOLATED FROM THE RECENTLY DISCOVERED
REMAINS OF THE RUSSIAN ROYAL FAMILY
ALEXEIS FACTOR VIII GENE WAS NORMAL!
ALEXEIS FACTOR IX GENE HAD A SINGLE
BASE MUTATION IN INTRON 3 THAT CREATED
A NEW SPLICE SITE
THIS PRODUCED A 2 bp FRAME SHIFT DISTAL
TO EXON 3 RESULTING IN PREMATURE CHAIN
TERMINATION OF FACTOR IX
THE ROYAL DISEASE WAS ACTUALLY
HEMOPHILIA B OR CHRISTMAS DISEASE !
END SIM 3-5