CLS 442 Lecture 2 Secondary Hemostasis

8 coagulation factors

• FII (prothrombin)

• FVII (stable factor or proconvertin)

• FIX (plasma thromboplastin component, Christimas factor)

• FX (stuart-power factor)

• FXI (plasma thromboplastin antecedent)
  FXII (Hageman factor)

• FXIII(plasma transglutaminase, fibrin-stabilizing factor)

• Prekaliokrien (Fletcher)

All coag factors are serine proteases except _____

FXIII

7 cofactor proteins

• HMWK (Fitzgerald)

• TF (FIII, tissue thromboplastin)

• FV (labile factor, proaccelerin)

• FVIII (labile factor, AHF A)

• PS

• Thrombomodulin

• PZ

What are these cofactors for?

• HMWK

• TF

• FV

• FVIII

• PS

• Thrombomodulin

• PZ

• HMWK- FXIIa & PK

• TF- FVIIa

• FV- FXa

• FVIII- FIXa

• PS- PCa

• Thrombomodulin- thrombin in PCa

• PZ- ZPI

Regulatory proteins(inhibitors) (4)

• AT

• PC

• Heparin co-factor II

• TFPI

Other coag factors (4)

• FI (fibrinogen)

• vWF

• Ca+ (FIV)

• Phospholipids (PF3)

Almost all coag factors are synthesized by _____ except _____ which is made by _____(stored in ……) and ____(stored in…..)

liver; vWF; ECs; Weibel-Palade granules; MKs; PLT alpha granules

What is the heaviest coag factor?

vWF (500-20000kd)

Coag factor with shortest half life

VII (3-6h)

Coag factor with longest half life

HMWK

____ is carried in the plasma as bound to vWF to maintain ___ hr half-life. If not bound it will only have ____ life span and is used by …….

VIII; 12; mins; Hemophilia A

This is crucial for coagulation complexes to bind to PLT or cell membrane phospholipids and is needed to trigger coag.

Ionized calcium

What is the function of coagulation proteins

• act one each other in cascade rxn

• each factor(serine proteases) activate the next and so on

• outcome= fibrinogen —→ stable fibrin clot

Vit. K-dependent coag proteins

• SCZ27910(prothrombin group)

• require vit K to become y-carboxylated(functional)

• have 10-12 glutamic acid resdues at N terminal

• SCZ27910 key for assembly of coag complexes to make thrombin

What are the 3 coagulation complexes?

• Complex 1: extrinsic tenase

• Complex 2 intrinsic tenase

• Complex 3: prothrombinase

Extrinsic tenase

• TF + VIIa+ Ca²⁺+ Pl

• activates X to Xa, and IX to IXa

• activates X mostly

Intrinsic tenase

• IXa + VIIIa + Pl (PLT surface) + Ca²⁺

• activates X to Xa

Prothrombinase

• Xa + Va + Pl + Ca²⁺

• Activates prothrombin to thrombin

what are the key factors in coagulation (severe bleeding in Hemophilia A and vWD)

FVIII & vWF

FVIII and FIX are encoded by …….

genes on X chromosome

FVIII is ……….. faster than other factors

degraded in stored plasma

_____ is bound to and carried by vWF in plasma

FVIII

what is vWF?

• multimeric, subunits of 240kd polymerized to 500-2000kd in MK and ECs, degraded to smaller multimers by ADAMTS13, in blood vessels with higher shear rate

• has receptors for PLTs and collagen (bridges PLTs to subendothelium during PLT adhesion)

• associated with ABO groups, O group has lower level

____ & ____ are APR (inflammation)

vWF; FVIII

ADAMTS13

a disintergrin and metallloprotease with a thrombospondin type 1 motif, member 13

what makes up the contact system/ contact factor complex

FXII, PK, HMWK

Contact system

• basis of the aPTT test

• FXII activated by negatively charged surfaces, FXIIa converts PK(bound to HMWK) to K, K activates FXII

• CFC activates FXI, in turn activates FIX

• KEY activator of FXI is thrombin made by extrinsic tenase

• def. on CSFs don’t cause bleeding, only prolongs aPTT

What is thrombin?

• Converted from prothrombin

• key serine protease is coag with multiple functions

• major function: cleaning FPA and FPB from fibrinogen= spontaneous polymerization, clot initiation

• activates FV, FVIII, and FXI(positive feedback)

Thrombin bound to ______ activates PC

thrombomodulin

Thrombin triggers ______ of PLTs

aggregation

Thrombin activates ______

TAFI

Thrombin activates what to stabilize the fibrin clot

FXIII

What is fibrinogen?

• primary substrate of thrombin

• RI is plasma 200--400mg/dL. an APR

• essential for PLT aggregation thru binding the GP IIb/IIIa receptor

• PLTs absorb it from plasm and store in a granules

What is FXIII

• aka plasma transglutaminase

• needed for stabilization

• def. doesn’t affect PT or PTT but leads to clinical bleeded

Coagulation Pathways (3)

• Extrinsic Pathway

• Intrinsic Pathway

• Common Pathway

Extrinsic pathway

• TF:FVII primary initiator of in vivo coagulation

• Rxn order: VII, X, V, prothrombin, & fibrinogen

• assessed by PT test

Intrinsic pathway

• Rxn order: XII, pre-K, HMWK, XI, IX, VIII, X, V, prothrombin, and fibrinogen

• assessed by aPTT test

Common pathway

• includes X, V, prothrombin, & fibrinogen

• assessed by PT, aPTT, & TT

In vivo

physiology= in body

In vitro

in lab

Initiation

• in vivo

• extrinsic tenase complex is main initiator of thrombin generation

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

• FVII:TF complex activates low lvl of FX and FIX, and small amount of thrombin(ignites more thrombin generation thru positive feedback, max 3%)

Propagation

• >95% thrombin is generated

• Rxns are on PLT membrane

• Large PLT aggregates, intrinsic tenase activates FX 50-100 fold higher, prothrombinase complex generates burst of thrombin

TFPI function

with Xa, binds TF:VIIa

Thrombomodulin function

EC surface receptor for thrombin

PC function

Serine protease

PS function

cofactor for PC

AT function

serpin

Heparin cofactor II

serpin

ZPI function

serpin

Fibrinolysis

• final stage of hemostatic activations(hrs after)

• fibrinolytic proteins bind to fibrin as it’s forming

• Plasminogen(prod. by liver)= main substrate

• ECs secrete TPA(more effective) & UPA

• TPA degrades fibrin-bound plasminogen to make plasmin

• Plasmin degrades fibrin(& FI) into several fragments (X,Y,D,E, and D-D)

D-D

fibrin derived and a diagnostic test for thrombosis and fibrinolysis

PAI-1

• main inhibitor

• binds to and inactivates TPA & UPA

• a serpin

• prod. mostly by ECs & MKs

a₂-antiplasmin

• prod. by liver

• binds to and inactivates free plasmin(not bound to fibrin

TAFI

• synthesized by liver

• activated by the thrombin-thrombomodulin complex

• blocks binding of TPA and plasminogen to fibrin and formation of plasmin

Trauma-induced bleeding disorder

• acquired

• systemic shock leading to acute reduction in ADAMTS13, coag factor activation, TF release, & hyperfibrinolysis

ADAMTS13 def. is the root cause of ______

TTP (thrombosis)

Liver disease

• acquired

• reduction of SCZ27910

• at start, FVII reduced, PT is very sensitive to low FVII, PT prolonged in mild liver disease(sensitive early marker)

Vit K deficiency and liver dx have the same effect on PT, how do you differentiate them?

FV level

In liver disease, thrombocytopenia is due to and results in what?

high sequestration by enlarged spleen, abnormal PLT function (low aggregation i. aggregometry)

DIC associated with liver dx is due to low prod. of what?

thrombin or PC and PS, amy be chronic compensated or acute uncompensated

Acute DIC shows what?

prolonged PT, PTT, TT, low fibrinogen, increased D-dimer test

Chronic compensated DIC shows what?

only D-dimer is increased

FI assay in liver dx interpretation

>400 mg/dL (elevated) in early, mild liver dx; <200 mg/dL in moderate to severe liver dx, which causes dysfibrinogenemia and hypofibrinogenmia

TT in liver dx interpretation

prolonged in dysfibrinogenmia, hypofibrinogenemia, elevated FDPs, and UFH

RT in liver dx interpretation

prolongeed in hypofibrinogenemia, signif. prolonged in dysfibrinogenemia, unaffected by heparin; rarely used

PT in liver dx interpretation

prolonged, even in mild liver dx, bc of des-y-carboxyl facotrs replacing norm. factors II, VII, & X

PTT in liver dx interpretation

mildly prolonged in severe liver dx bc of DIC or des-y-factors II, IX, X

FV assay in liver dx interpretation

FV reduced in liver dx but is unaffected by vit k def., so FV level helps distinguish liver dx from vit k def.

PLT count in liver dx interpretation

mild thrombocytopenia, <150,000/µL

PLT aggregometry in liver dx interpretation

mild suppression of PLT aggregation and secretion in response to most agonists

Quantitative D-dimer in liver dx interpretation

>240 ng/mL or >500 ng/mL FEUs

Autoanti-FVIII inhibitor and acquired hemophilia

• Gene mutation

• Some patients(>65 yrs old) may develop auto AB against FVIII which causes an acquired hemophilia condition(sudden bleeding into soft tissue of GI bleeding

• may develop in autoimmune diseases like RA, IBD, SLE, or lymphoproliferative disease(CLL)

• could be fatal in 20% cases even if treated

Lab investigation of FVIII inhibitors

• PT, PTT, TT for anyone experiencing sudden anatomic hemorrhage that mimics acquired hemophilia(no fam history or disease in background)

• PPT prolonged; PT & TT are normal

• FVIII level <40 units/dL should be established by factor assay

what occurs in mixing studies to confirm presence of FVIII inhibitor

• PP mixed with 1:1 NP, prolonged PTT will be corrected initially but prolonged again after incubation in 37C dude to AB usually Igg or temp dependent

• If prolonged PTT remains even after 2h incubation, indicates= lupus anticoagulants or heparin therapy

• 15% of inhibitors(high avidity) immediately prolongs PPT’s mixture

vWD

• congenital

• both sexes but F>M

• quali. or quanti. changes in vWF due to gene mutations

• most common inherited mucocutaneous bleeding disorder causing decreased PLT adhesion and impaired primary hemostasis

• Severe quanti. vWF def. causes FVIII def.

• in low vWF cases, vWF plasma lvl (30-50%) maintain sufficient FVIII

vWD types & subtypes (6)

• type 1 - quanti. 40-70%, mild to mod

• type 2- qualit. types

  • type 2A

  • type 2B

  • type 2M

  • type 2N

• type 3- null phenotype, severe vWF def, very low FVIII

vWD lab diagnosis

• Very helpful: history of mucocutaneous bleeding and decreased vWF conc. or activity

• CBC to rule out thrombocytopenia

• PT and PTT to rule out factor def. other than vWF

what is the standard vWD test panel (6)?

• vWF: Rco

• vWF: Ag

• VWF activity to VWF: AG ratio

• RIPA

• FVIII activity

• vWF multimers

in VWD, thrombocytopenia happens only in……?

subtype 2B

VWD Type 1 diagnosis?

VWF multimers: normal pattern only

VWD subtype 2B diagnosis?

• PLT count: Decreased

• RIPA: Increased

VWD subtype 2M diagnosis?

• PLT count: decreased

• VWF multimers: normal pattern

VWD subtype 2N diagnosis?

• VWF:RCo: Normal

• VWF multimers: Normal pattern

VWD type 3 diagnosis?

• RIPA: absent

• FVIII activity: <10 units/dL

• VWF multimers: All forms absent

Hemophilia A & B

• congenital

• single factor def.

• marked by anatomic soft tissue bleeding

Hemophilias A=

FVIII def. (85% of patients)

Hemophilia B=

FIX def. (14% of patients)

Hemophilia C=

FXI def. (1% of patients)

What is hemophilia A?

• FVIII protein translated from X chromosome

  • various deletions, stop codons, nonsense and/or missense mutations result in quantitative def. of FVIII

• FVIII def, significantly slows coag pathway prod. of Thrombin= bleeding

What are the clinical manifestations of Hemophilia A (5)?

• anatomic bleeds

• deep muscle and joint hemorrhages

• hematomas

• wound oozing after trauma or surgery

• bleeding into CNS, peritoneum, GI, and kidneys

FI: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Pro; Pro; Pro; Fibrinogen assay

FII: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Pro; Pro; Norm; Prothrombin, V,VII,X assays

FV: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Pro; Pro; Norm; Prothrombin, V, VII, X assays

FVII: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Pro; norm; norm; VII assay

FVIII: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Norm; pro; norm; VIII, IX, XI assays

FIX: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

Norm; pro; norm; VIII, IX, XI assays

FX: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

pro; pro; norm; prothrombin, V, VII, X assays

FXI: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

norm; pro; norm; VIII,IX,XI assays

FXIII: PT, PTT, TT, & Reflex Test results of Clot-Based Assays in Congenital Single-Factor Def.

norm; norm; norm; XIII quanti. assay