Lecture 2 - Hemostatis part 2

8 factors are zymogens

circulating in plasma in an inactive form

Prothrombin

Stable factor or proconvertin

Plasma thromboplastin component, Christmas factor

Stuart-Prower Factor

Plasma thromboplastin antecedent

Hageman Factor

plasma transglutaminase, fibrin-stabilizing factor

Fletcher

ALL except FXIII (13)

7 proteins

stabilize and accelerate proteolytic reactions

HMWK, Fitzgerald
Tissue Factor (FIII, tissue thromboplastin)
FV(liabile factor, proaccelerin)
FVIII (liable factor, AHF A)
Protein S (co-factor for activated protein C)
Protein Z (co-facotor for protein Z-dependent protease inhibitor, ZPI)
Thrombomodulin (Co-factor for thrombin in protein C activation

High molecular weight kininogen

Anti-thrombin
Protein C
Heparin
Co-factor II
TFPI (tissue factor pathway inhibitor)

FI (fibrinogen), vWF, Ca+ (FIV), phospholipids (PF3)

"active or activated" (e.g., VIIa, XIIa

order of discovery or description of factors

Fibrinogen

Prothrombin

PK (prekallikren) and HMWK (high molecular weight kininogen)

Almost all except vWF

vWF (500-20,000 kD)

FVII (7) ; 3-6 hrs <- shortest
HMWK <- longest

in plasma as bound to vWF to maintain 12 hr halflife

Ionized calcium!! to bind to platelet or cell membrane phospholipids.

each factor (serine protease) activates the next, and this continues...

conversion of fibrinogen (ultimate substrate) into stable fibrin clot

SCZ27910 (aka a prothrombin group reagent)

require vit K to become γ-carboxylated (hence functional)

10-12 glutamic acid residues at N terminal

3

COMPLEX 1: EXTRINSIC TENASE
COMPLEX 2: INTRINSIC TENASE
COMPLEX 3: PROTHROMBINASE

Tissue factor + VIIa + Ca2++ phospholipid;
it activates X to Xa, and IX to IXa

IXa (9)+ VIIIa (8)+ phospholipid (PLT surface) + Ca2+ ;
Activates X (10) to Xa

Xa (10) + Va (5) + phospholipid + Ca2+ ;
Activates prothrombin to thrombin

FVIII (8)and vWF

X chromosome

vWF

multimeric, subunits of 240 kd polymerized to 500 -2000kd in MK and ECs, degraded to smaller mulitmers by ADAMTS13 (a disintergrin and metalloprotease with a thrombospondin type 1 motif, member 13) in blood vessels with higher shear rate

PLTs and collagen; bridges PLTs to subendothelium during PLT adhesion

ABO groups, O group has lower level.

vWF and FVIII (8)

FXII (12), PK, HMWK

- FXIIa converts PK (bound of HMWK) to K, K further activates FXII (12)
- CONTACT FACTOR COMPLEX (HMWK + PK + FXIIa) activates FXI ( in turn activates FIX (9)

THROMBIN!! ; generated by the extrinsic tenase

NO bleeding BUT do prolong aPTT.

- KEY serine protease in coagulation with multiple functions;
- Cleaving fibrinopeptides A and B (FPA and FPB) from fibrinogen, triggering spontaneous polymerization, clot intitiation.

FV (5), FVIII (8), AND FXI (11)( postiive feedback loop)

A: It cleaves fibrinopeptides A and B from fibrinogen, turning it into fibrin to start clot formation.
A: Factors V, VIII, and XI — a positive feedback loop.
A: It activates protein C, which turns off clotting (anticoagulant function).
A: It activates platelets, helping them stick and form a plug.
A: Thrombin activates TAFI (thrombin activatable fibrinolysis inhibitor), which slows down fibrinolysis (clot breakdown).
A: It activates FXIII, which cross-links fibrin and stabilizes the clot.

primary substrate of thrombin

200-400mg/dL, an APR

aggregation; GPIIb/IIa

PLTs absorb it and stor in alpha granules

plasma transglutaminase aka a fibrin stabilizing factor

deficiency does not affect PT or PTT BUT leads to clinical bleeding

FVII (7) primary initiator of in vivo coagulation

VII, X, V, prothrombin, and fibrinogen

Prothrombin Time (PT) test

XII, pre-K, HMWK, XI, IX, VIII, X, V, prothrombin, and fibrinogen

activated partial thromboplastin time (aPTT) test

X, V, PROTHROMBIN AND FIBRINOGEN

Both tests plus thrombin test (TT)

endothelial

Initiation and Propagation

1-2% FVIIa circulates but inert (requires TF)

a complex that can turn on other clotting factors (like Factor X (10) and IX (9)) <-low levels;
This creates a small amount of thrombin which is just enough to start a chain reaction (positive feedback loop) that makes more thrombin but still just a little at this stage (about 3% max)

>95% thrombin is generated.;
happens on the PLT membrane.

When lots of platelets stick togehter (form aggregates), they provide a perfect surface for the Intrinsic tenase complex. ;
this super-activates Factor X (FX) - up to 50 to 100 times more than in the initiation phase.

this leads to the formation of the prothrombinase complex, which causes a big burst of thrombin - enough to really build a strong clot.

Anti-thrombin
Protein C
Heparin Cofactor II
TFPI (tissue factor pathway inhibitor)

A1: With Xa, it binds TF:VIIa.

A2: It is an EC surface receptor for thrombin.

A3: Serine protease.

A4: Cofactor.

A5: Serpin.

A6: Serpin.

A7: Serpin.

Final stage of hemostatic activation (hours after)

Main substrate is plasminogen produced by the liver

A: TPA (tissue plasminogen activator) and UPA (urokinase plasminogen activator); TPA is more effective.

A: It degrades fibrin-bound plasminogen to generate plasmin.

A: It degrades fibrin and fibrinogen into smaller fragments: X, Y, D, E, and D-D.

A: D-dimer is fibrin-derived and is used as a diagnostic test for thrombosis and fibrinolysis.

A: Plasminogen activator inhibitor-1 (PAI-1), which inactivates TPA and UPA.

A: It’s a serpin (serine protease inhibitor), mostly produced by endothelial cells (ECs) and megakaryocytes (MKs).

A: It binds to and inactivates free plasmin, not plasmin that's bound to fibrin.

A: In the liver.

A: The thrombin-thrombomodulin complex.

A: It blocks TPA and plasminogen from binding to fibrin and prevents the formation of plasmin.

Trauma or liver disease

- A drop in ADAMTS13 (a protein that controls clotting)
- Activation of clotting factors
- Release of tissue factor
- Hyperfibrinolysis (breaking down clots too much)

bleeding can happen.

SCZ27910 levels drop in liver disease ( this protein helps with clotting)
- The liver also makes Factor VII (7), which has the shortest half-life, so it drops first.
- Since PT (prothrombin time) is very senstitive to FVII (7) levels, PT becomes PROLONGED early in liver disease -- A USEFULL EARLY WARNING SIGN!
- Vitamin K deficiency also prolongs PT (because it's needed to make FVII (7).

Doctors look at Factor V (5);
- FV is normal in vitamin K deficiency
- FV is low in liver disease

1. Low Platelets (Thrombocytopenia)
2. DIC can happen in Liver Disease
3. Acute DIC signs
4. Chronic (compensated) DIC

- An enlarged spleen (common in liver disease) traps and destroys platelets.
- Platelets that are present don't work properly bc of it-- they don't stick together well (shown by low results in platelet aggregation tests)

DIC = Disseminated Intravascular Coagulation= body uses up clotting factors too fast
- Liver makes natural clot blockers like antithrombin or protein C, and protein S.
- If the liver is damaged, it doesn't make enough, leading to unregulated clotting (DIC).
- This can be chronic /(body adjusts) or acute (sudden and dangerous)

Long clotting times: PT, PTT, and TT are all prolonged
Low fibrinogen ( used up from too much clotting)
High D-dimer test (shoes fibrin is being broken down a lot)

the body adjusts over time ;
ONLY D-dimer is increased , other labs might look normal.

->400 mg/dL (elevated) in early, mild liver disease
<200 mg/dL in moderate to severe liver disease, which causes dysfibrinogenemia
and hypofibrinogenemia

Dysfibrinogenemia: Normal amount of fibrinogen is made, but it doesn’t work properly — it's defective and can’t help form clots.

Hypofibrinogenemia: The body doesn’t make enough fibrinogen, leading to a shortage that affects clot formation.

It suggests dysfibrinogenemia, hypofibrinogenemia, or elevated fibrin degradation products; can also be affected by heparin.

It’s not affected by heparin and is prolonged in dysfibrinogenemia or hypofibrinogenemia.

Prothrombin time (PT) — because of reduced production of vitamin K–dependent factors like prothrombin (II), VII, and X.

A: In severe liver disease due to loss of clotting factors or DIC.
Mildly prolonged in severe liver disease because of disseminated
intravascular coagulation (DIC) or des-γ-carboxyl factors II
(prothrombin), IX, and X

A: Factor V is low in liver disease but normal in vitamin K deficiency.

A: Mild thrombocytopenia with a platelet count less than 150,000/μL.

Mild suppression of platelet aggregation and secretion in response
to most agonists

A: >240 ng/mL or >500 ng/mL FEUs (fibrinogen equivalent units)

A: Autoantibodies against Factor VIII (8) that inhibit its function.

A: Patients over 65 years old.

A: Sudden bleeding into soft tissue or the gastrointestinal (GI) tract.

Rheumatoid arthritis (RA), inflammatory bowel disease (IBD), lupus (SLE), and chronic lymphocytic leukemia (CLL).

A: Up to 20%.

A: PT, PTT, and TT.

A: Prolonged PTT, normal PT and TT.

A: A FVIII level below 40 units/dL, confirmed by factor assay.

A: It suggests a time-dependent inhibitor, like anti-FVIII.

A: The patient may have lupus anticoagulants or is on heparin therapy.

A: The patient has a high-avidity inhibitor (present in ~15% of cases).

genetic bleeding disorder caused by a problem with von Willebrand factor (vWF), a protein that helps platelets stick and protects Factor VIII.

A: It is autosomal dominant, affects both sexes, but symptoms are more common in females.

A: Gene mutations that lead to either too little (quantitative) or abnormal (qualitative) vWF.

A: It’s the most common inherited mucocutaneous bleeding disorder that impairs platelet adhesion and primary hemostasis.

A: Severe deficiency of vWF leads to Factor VIII deficiency, worsening the bleeding risk.

A: vWF levels are around 30–50%, which is usually enough to maintain normal Factor VIII levels.

A: Type 1 — a quantitative deficiency found in 40–70% of cases; usually mild to moderate.

A: It is a qualitative disorder, meaning vWF is present but doesn’t function properly.

A: A subtype of Type 2 where large vWF multimers are missing, leading to poor platelet adhesion.

A: A subtype where vWF has increased affinity for platelets, causing them to be cleared from circulation and possibly leading to thrombocytopenia.

A: A qualitative defect where vWF has reduced platelet binding, but multimers are normal.

A: A subtype with defective binding of vWF to Factor VIII, leading to low FVIII levels, mimicking mild hemophilia A.

A: A null phenotype — almost no vWF is produced, causing severe bleeding and very low Factor VIII levels.

A: History of mucocutaneous bleeding and decreased vWF concentration or activity.

A: To rule out thrombocytopenia (low platelet count) as a cause of bleeding.

A: To rule out deficiencies in other clotting factors unrelated to vWF.

A: It measures vWF activity — how well vWF helps platelets stick together.

A: The amount of von Willebrand factor in the blood.

A: Whether the vWF is functioning properly — a low ratio suggests dysfunctional vWF.

A: To help detect vWD Type 2B, which causes abnormal platelet aggregation.

A: Because vWF protects Factor VIII, so low vWF may lead to low FVIII activity.

A: To evaluate the structure and size of vWF — useful for identifying vWD subtypes.

A: Both are low due to a quantitative deficiency of vWF.

A: Large and intermediate multimers are absent, affecting vWF function.

A: Type 2B — due to increased binding of vWF to platelets, causing platelet clearance.

A: vWF:Ag and vWF multimers are normal, but vWF activity is low.

A: Type 2N — vWF can't bind/stabilize FVIII.

A: Very low/absent vWF and FVIII, prolonged PTT, absent RIPA (ristocetin-induced platelet aggregation), and no multimers.

A: It is normal to slightly prolonged.

A: vWF is normal, but Factor VIII is low.

X-linked recessive: lack of a single clotting factor, which causes bleeding into muscles, joints, or soft tissue (not sure surface bleeding like in vWD.
- Males (who have only one X) usually have the disease.
- Females (who have two X chromosomes) are usually carriers and may not show symptoms.

3:
Hemophilia A, B, C

Missing Factor VIII (most common - 85% of cases)

Missing Factor IX (about 14% of cases)

Missing Factor XI (very rare - about 1% of cases, and not X-linked)

A: A congenital single-factor deficiency, meaning a person is born missing one clotting factor.

A: Anatomic soft tissue bleeding, such as into joints and muscles.

A: X-linked recessive — males are affected, females are typically carriers.

A: Factor VIII (FVIII) — it accounts for 85% of hemophilia cases.

A: Factor IX (FIX) — it causes about 14% of hemophilia cases.

A: Factor XI (FXI) — very rare, found in only 1% of cases.

A: On the X chromosome.

A: It slows down thrombin production, which is essential for clotting, leading to increased bleeding.

Anatomic bleeds

Deep muscle and joint hemorrhages

Hematomas (deep bruises)

Wound oozing after injury or surgery

Internal bleeding in the CNS, GI tract, kidneys, and more

You should consider testing Factor XIII, because:

Factor XIII deficiency causes normal PT, PTT, and TT,

But patients still bleed, especially after trauma or surgery.

It's not detected by routine clotting tests — so you need a Factor XIII quantitative assay to diagnose it.

A: To tell the difference between factor deficiencies and factor inhibitors.

Factor deficiencies (single or multiple)

Specific factor antibodies or inhibitors

Non-specific antibodies or inhibitors

A: At least 50% of the normal level.

A: They help decide the next steps to find the cause of abnormal PT or APTT.

A: It suggests a clotting factor deficiency.

A: It suggests the presence of an inhibitor, like a factor antibody.

A: It indicates a time-dependent inhibitor, such as an anti-Factor VIII antibody.

Procedure:
1. Repeat PT and/or APTT using patient PPP (platelet poor plasma)
2. Run Normal Pooled Plasma alone
• Ensure normal results or 100% of factor level
3. Run patient PPP mixed 1:1 with NPP
4. Compare results
• Initial testing is compared to mix
• Did mix correct? Did mix not correct?

A: An inhibitor assay, such as DRVVT (dilute Russell viper venom time).

A: Incubate the mix for 1–2 hours at 37°C to distinguish between a factor deficiency and a factor antibody.

A: Factor deficiency.

A: Presence of a factor-specific antibody.

A: Factor antibody assays.

A: It suggests the presence of an inhibitor, such as lupus anticoagulant.

A: To specifically assess factor XIII (FXIII) deficiency or inhibition.

A: FXIII is insoluble in urea.

A: The fibrin clot will dissolve rapidly.

A: The result should be confirmed by an FXIII assay.

A: A quantitative FXIII activity assay.

A1: The time it takes for a fibrin clot to form after thrombin is added to plasma.

A2: A commercially available bovine thrombin reagent.

A3: 37°C for 3 minutes.

A4: 200 µL.

A5: 15–20 seconds.

A6: Hypofibrinogenemia, afibrinogenemia, or dysfibrinogenemia.

A7: The Reptilase time test.

A1: It measures how long it takes plasma to clot via the extrinsic and common coagulation pathways.

A2: 37°C.

A3: 3–10 minutes.

A4: Coagulation factors may deteriorate or be affected by pH changes and evaporation.

A5: Thromboplastin (tissue factor), phospholipids, and calcium.

A6: A timer is started, and time to clot formation is recorded.

A7: Visually or with optical/electromechanical sensors.

A8: 9–15 seconds.

A1: It was developed to standardize PT reporting across labs due to variations in thromboplastin reagents.

A2: It standardizes PT results and reporting, making it possible to monitor Vitamin K antagonist therapy across different labs.

A3: Warfarin or Coumadin.

A4: INR is reported alongside the PT result.

A1: PT (in seconds) and ISI are used to calculate the INR (International Normalized Ratio).

A2: ISI stands for International Sensitivity Index.

A3: Commercial suppliers provide the ISI value with each thromboplastin reagent lot.

A4: It indicates how sensitive the reagent is to deficiencies in Vitamin K–dependent factors compared to the WHO reference standard.

10.6 – 14.6 seconds

9.5 – 13.5 seconds

0.9-1.2

Because they can vary from lab to lab.

Once a year.

PT is reported in seconds and INR is calculated manually.

They automatically calculate the INR.

Activated Partial Thromboplastin Time; it is a clot-based routine coagulation test.

APTT is used to assess deficiencies or inhibitors of the factors in the intrinsic and common pathways.

Factors XII (12), XI (11), IX (9), VIII (8), X (10), V (5), II (prothrombin), and I (fibrinogen).

Factor VII is not assessed by APTT.

Standard (unfractionated) Heparin anticoagulant therapy.

3.2% sodium citrate blue top tube;
leaves all clotting factors available except
Calcium, Citrate binds the calcium in the plasma and
therefore prevents coagulation, but not as strongly as EDTA

Plasma contains coagulation factors.

Plasma is separated from platelets by centrifugation. at 3000 RPM for 15 min at room temperature

Platelet Poor Plasma (PPP) is plasma with a platelet count less than 10x10^9/L.

9:1 blood-to-anticoagulant ratio is required for optimum
results (9 parts whole blood to 1 part sodium citrate)

• High Hematocrit (≥ 55%) affects ratio