Apheresis & Anticoagulation

2019-מרץ-26

ר אשר וינדר"ד

מנהל מכון המטולוגי

מרכז רפואי וולפסון

APHERESIS

2019

Apheresis & Anticoagulation

• What is hemostasis

• Components & Stages of hemostasis

• How to measure hemostasis

• Antithrombotic drugs

• Anticoagulants & reversal agents

– Heparin & LMWH

– Warfarin

– DOACs

– Citrate

What is hemostasis?

ThrombosisBleeding

Hemostasis

Vessel Wall

Clotting

FactorsPlatelets

Vascular integrityP

late

let

reac

tion

Coagulation cascade

Hemostasis

Schematic of in vivo coagulation modelReactions of degradation (reactions 59–105) are not shown

ATIII, antithrombin III; APC, activated

protein C; CA, contact activator;

FDP, fibrin degradation product; Fg,

fibrinogen; FIIa, activated FII; FIXa,

activated FIX; FVa, activated FV;

FVIIa, activated FVII; FXa, activated

FX; FXIa, activated FXI; FXIIa,

activated FXII; FXIIIa, activated

FXIII; K, kallikrein; mIIa,

meizothrombin; P, plasmin; PC,

protein C; Pg, plasminogen; TF,

tissue factor; TFPI, tissue factor

pathway inhibitor; Tmod,

thrombomodulin; t-PA, tissue-type

plasminogen activator.Lee D, Nayak S, Martin SW, et al. J Thromb Haemost 2016; 14: 2430–45

Coagulation cascade

Macfarlane RG. Nature 1964; 202: 498-499.

Fibrin clot

Secondary Hemostasis - Coagulation system

Normal fibrin network

Fibrinolysis

Prothrombin

Plasminogen

COAGULATION

CASCADE

Platelets

Thrombin

Fibrin

ClotFibrinogen

XII XIIa

XI XIa

IX IXa

VIIIa

XVIIa

TF

VaXa

PlasminPA

FIBRINOLYSIS

VWF

Clotting time

Whole blood 4-8 min

Whole blood + EDTA or citrate infinite

Citrated platelet-poor plasma + Ca++ 2-4 min

Citrated platelet-poor plasma + PL + Ca++ 60-85 sec

Citrated platelet-poor plasma + kaolin + PL + Ca++ 21-32 sec (aPTT)

Citrated platelet-poor plasma + thromboplastin + Ca++ 11-12 sec (PT)

Coagulation in vitro

Addition of EDTA or citrate prevents clotting by binding calciumkaolin = insoluble aluminum silicatethromboplastin = a saline brain extract containing tissue factor

Prothrombin time (PT)

Blood sample + Citrate

Tissue Thromboplastin

Mix with phospholipid extract

Add calcium and blood sample

Determine clotting time

Generally 12 - 14 seconds

Used to detect defects in extrinsic pathway

Procedure (1)

Procedure (2)Reference range = 10.5 - 13.5 sec (STH)

ThrombinProthrombin

Ca2+

FXa

Prothrombinase

FVa HC

FVa LC

Ca2+

In-vivo vs Ex-vivoelectromechanical measuring principle

Antithrombotic Agents:

Mechanism of Action

• Antiplatelet drugs: interfere with platelet activity

• Anticoagulants: prevent clot formation and extension

• Thrombolytic agents: dissolve existing thrombi

Thinning The Blood?

• Anticoagulation is a term which means "reducing

the bloods potential to clot".

• Many people interchange the expression

"thinning of the blood" with the term

"anticoagulation"

The (Good) Old Days

• Aspirin

• Heparin

• Warfarin (Coumadin)

Antithrombotic Therapy – 2019Approved by Israeli MoH

Antiplatalet

• Aspirin

• ADP receptor inhibitors -Thienopyridines

– Clopidrogel

– Prasugrel (Effient)

– Ticagrelor (Brilinta)

– Ticlopidine

• Dipyridamol (Aggrenox)

• GPIIb-IIIa inhibitors

– Abciximab (ReoPro)

– Tirofiban (Aggrastat)

– Eptifibatide (Integrilin)

Anticoagulants

• Heparins– UFH– LMWH

• Danaparoid

• Coumadin, Sintrom

• Bivalirudin (Angiomax)

• Fondaparinux (Arixtra)

• Rivaroxaban (Xarelto)

• Dabigatran (Pradaxa)

• Apixaban (Eliqius)

Fibrinolytics

• SK

• U-PA

• rt-PA

1 Amediplase

2 Alfimeprase

3 Desmoteplase

4 AZD-9684

5 V-10153

6 Microplasmin

7 HTU-PA (Hybrid-B PA)

8 PAI-749

Thrombolytics

1 Prasugrel (CS-747)

2 Ecraprost

3 S-18886

4 NCX-4016

5 AZD-6140

6 Cangrelor

7 NM-702

8 Liprostin

9 INS-50589

10 CLB-1309

11 Xemilofiban

12 SL-650472

Antiplatelets

Phase III

Phase II

Phase I

Anticoagulants Thrombolytics Antiplatelets1 Idraparinux

2 Dabigatran

3 ART-123

4 Tifacogin

6 SR-123781

7 AZD-0837

8 MCC-977

9 SSR-182289

10 TGN-255

11 Odiparcil

12 TTP-889

13 rNAPc2

14 YM-150

15 DX-9065a

16 Rivaroxaban

17 XRP-0673 (otamixaban)

18 Apixaban (BMS)

19 LY-517717

20 DU-176b

21 TGN-167

22 AVE-5026

23 SCH-530348

24 KFA-1982

25 EMD-503962

26 SSR-126517

27 Oral heparins

28 ARC-183

Anticoagulants

The antithrombotic pipeline is heavy of novel agents

l

1 2

7

5

1

6

811

10 9

8 7

6

5

4

3 2

12

4

3

12 3

4 5y

8y y7

9 10

1y2 11y13

1y4 15

18y

6

19

2122

2324

26

272829

17

25

16y

20

Oral Oral

Parenteral Parenteral

Oral

Heparin

Heparin is a heterodispersed mixture of a complexnegatively-charged glycosaminoglycan (that is,polysaccharide) with a Mr that range from 3,000 to40,000 daltons.

Coagulation cascade and the use of

heparin

Heparin mechanism of action

Heparin

Antithrombin IIIThrombin

Heparan sulfate anticoagulant

pathway

UFH

LMWHs

depolymerization

Low LMWHs

Ultra low LMWHs

Depolymerization

1. Chemical

2. Enzymatic

3. Physical

Advantages of LMWH over unfractionated heparin

More reliable relation between dose and response

Does not need monitoring

Long duration of action

Does not need dose adjustments

Lower incidence of thrombocytopenia

Can be administered by patient at home

Saves about five to six days' admission per patient

Reversal of HeparinProtamine sulfate

• a polycationic, highly positively charged protein derived from salmon sperm protein

• The mechanism of action of protamine involves binding to the negatively charged heparin molecules, which forms a stable complex

• Immediate reversal of heparin requires 1 mg of protamine for every 100 U of UFH administered within the last 2–3 hours.

• LMWH has a longer half-life compared to UFH and is only partially reversed by protamine (about 60%).

• Risks: systemic hypotension and bradycardia, cases of noncardiogenic pulmonary edema within minutes to 1 or 2 hours after the administration of the drug, frequently associated with an anaphylactic reaction and bronchospasm

Vitamin K

Synthesis of Functional

Coagulation Factors

VII

IX

X

II

Vitamin K-Dependent Clotting

Factors

Warfarin Mechanism of Action

Warfarin

Synthesis of non functional

coagulation factors

Warfarin Mechanism of Action

Vitamin K

VII

IX

X

II

Warfarin prevents the reduction of vitamin K epoxides in liver microsomes and induces a state analogous to vitamin K deficiency.

Fibrin Clot

XIICoagulation Cascade

Vitamin K Dependent Factors

Intrinsic

ExtrinsicXI

IX

VIII VII

X

V

II

I

6 hr

36 hr

60 hr

24 hr Tissue Factor

PT / INRaPTT

J Clin Path 1985; 38:133-134; WHO Tech Rep Ser. #687 983.

INR: International Normalized Ratio

• A mathematical “correction” (of the PT ratio) for

differences in the sensitivity of thromboplastin

reagents

• INR is the PT ratio one would have obtained if the

“reference” thromboplastin had been used

• Allows for comparison of results between labs and

standardizes reporting of the prothrombin time

( )Patient’s PT in Seconds

Mean Normal PT in SecondsINR =

ISI

ISI = International Sensitivity Index

MeanNormal(Seconds)

PTR ISI INR

12

12

13

11

14.5

1.3

1.5

1.6

2.2

2.6

A

B

C

D

E

Blood from a single patient Patient’s

PT(Seconds)

16

18

21

24

38

ThromboplastinReagent

How Different Thromboplastins

Influence the PT Ratio and INR

MeanNormal(Seconds)

PTR ISI INR

12

12

13

11

14.5

1.3

1.5

1.6

2.2

2.6

3.2

2.4

2.0

1.2

1.0

2.6

2.6

2.6

2.6

2.6

A

B

C

D

E

Blood from a single patient Patient’s

PT(Seconds)

16

18

21

24

38

Thromboplastinreagent

How Different Thromboplastins

Influence the PT Ratio and INR

Intensity of Anticoagulation (INR)

Clin

ical

Eve

nts

Therapeutic

Window

Indication INR Range Target

Prophylaxis of venous thrombosis (high-risk surgery) 2.0–3.0 2.5

Treatment of venous thrombosis

Treatment of PE

Prevention of systemic embolism

Tissue heart valves

AMI (to prevent systemic embolism)

Valvular heart disease

Atrial fibrillation

Mechanical prosthetic valves (high risk) 2.5–3.5 3.0

Certain patients with thrombosis and the antiphospholipid syndrome

Bileaflet mechanical valve in aortic position, NSR 2.0–3.0 2.5

Warfarin: Current

Indications/Intensity

Distribution of time in therapeutic range among incident atrial fibrillation patients treated with

warfarin - ISRAEL

Moti Haim et al. J Am Heart Assoc 2015;4:e001486

© 2015 Moti Haim et al.

Individual Time in Therapeutic Range

(TTR) among warfarin users was 42%.

Reversal agents for VKA associated anticoagulation

Quinlan DJ, et al. Circulation 2013;128:1179-81. Goldstein JN, et al. Lancet 2015;385:2077-87

4F-PCC vs

FFP for rapid

VKA reversal

INR ≤ 1.3 at 0.5 hours after the end of infusion

Managing Patients with High INR ValuesIntravenous or oral phytonadione (vitamin K1)

Arch Intern Med. 2003;163:2469-2473

A: patients with baseline INR of 6 to 10 or B: INR greater than 10. Data are presented as mean ± SD.

Composition of Beriplex P/NBeriplex® P/N 250content per vial

Beriplex® P/N 500content per vial

Total protein 60–140 mg 120–280 mg

Human plasma fraction (activity)

Factor II 200–480 IU 400–960 IU

Factor VII 100–250 IU 200–500 IU

Factor IX 200–310 IU 400–620 IU

Factor X 220–600 IU 440–1200 IU

Protein C 150–450 IU 300–900 IU

Protein S 120–380 IU 240–760 IU

Other ingredients include small quantities of antithrombin III and heparin

VIIa

Xa

IXa

XIa

XIIa

Direct oral anticoagulants (DOACs)

Tissue

factor

Dabigatran

II×Rivaroxaban

Apixaban

Edoxaban

×Fibrinogen Fibrin clot

DabigatranRivaroxabanApixabanהשם הגנרי

®PRADAXA®XARELTO®ELIQUISהשם המסחרי

Direct thrombinמנגנון פעולה

inhibitor, reversible

Factor Xa inhibitor,

reversible

Factor Xa inhibitor,

reversible

Pro-drugלאלאכן

3-7%זמינות ביולוגיתקיבה ריקה66%

עם אוכל~ 90%50%

12-145-138-14(שעות)זמן מחצית חיים

כבדיכבדיכבדימטבוליזם

CYP3A4/5י "בעיקר עCYP3A4/5י "בעיקר עלאCYPמטבוליזם

כןכןכןP-gpסובסטרט של

80%33%27%פינוי כלייתי%

נדרשתנדרשתנדרשתכ "התאמת מינון באס

אין השפעה 40%-מעלה ספיגה בכאין השפעה השפעה למזון

אין חשיבותעם האוכל אין חשיבותנטילה ביחס למזון

פעמיים ביום פעם ביוםפעמיים ביום תדירות מתן

IdarucizumabPraaxbindTarget: anti IIa

Phase IIIPhase IIPhase Iapproved byFDA & EMA

Andexanet alfaAndexxaTarget: anti Xa

Phase IIIPhase IIPhase I

Ciraparantag(PER977)Target: global ?

Phase IIPhase I

approved byFDA

Development of DOAC reversal agents

Idarucizumab for the reversal of dabigatran

Schiele et al. Blood 2013; Glund et al. Thromb Haemost 2015; Eikelboom et al. Circulation 2015

IdarucizumabDabigatran

Thrombin

• Humanized Fab: binds free dabigatran and dabigatran bound to thrombin

• Binding affinity for dabigatran ~350× higher than dabigatran for thrombin

• IV administration, immediate onset of action

• No intrinsic procoagulant or anticoagulant activity

• Short half-life: Initial: ~45 min, Terminal: 4.5–8.1 hrs

Dabigatran + placebo (n=9)Dabigatran + 4 g idarucizumab (day 4) (n=8)

Normal upper reference limit (n=86)Mean baseline (n=86)

–2

Time after end of infusion (h)

dT

T (

s)

70

65

60

55

50

45

40

35

30

0 2 4 6 8 10 12 24 36 48 7260

End of idarucizumab

(5-min infusion)

Idarucizumab: reversal of dabigatran

anticoagulation in volunteers

Management of rivaroxaban- or apixaban-associated major bleeding with prothrombin complex concentrates: a cohort studyOutcome of bleeding with PCC

Majeed A., et al. Blood 2017 130:1706-1712.

PCC was given at a median (interquartile range) dose of 2000 IUManagement with PCC was assessed as effective in 69% of patients and ineffective in 31%

Andexanet Alfa for the Reversal of Factor Xa

Lu, G. et al. Nat. Med 2013;19, 446–451; Siegal DM, et al. N Engl J Med 2015; 373:2413-2424

modified recombinant

enzymatically inactive

factor Xa that traps

circulating Xa inhibitor

Healthy volunteer with an

age range of 50 to 75 years

Ciraparantag (Aripazine, PER977)

binds to unfractionated heparin, low-molecular-weight heparin, fondaparinux, dabigatran, rivaroxaban, apixaban and edoxaban through hydrogen bonding and charge–charge interactions

a small, synthetic, water-soluble, cationic molecule, intravenously administered

Trisodium citrate (Sodium citrate)

• A citrate is a derivative of citric acid; that

is, the salts, esters, and the polyatomic

anion found in solution.

• Trisodium citrate is the salt of citric acid

Na3C6H5O7

Applications

• Foods: a food additive for flavor or as a preservative: club soda, bratwurst, gelatin mix, ice cream, yogurt, jams, sweets, milk powder, processed cheeses, wine…

• Buffer: As a conjugate base of a weak acid: gelatin desserts, antacids, such as Alka-Seltzer

• Anticoagulant:– blood transfusions / apheresis bags (CPD, ACD) – in blood-collection tubes. – locking agent in vascular catheters and – hemodialysis lines instead of heparin

Trisodium citrate in Blood

• tri-sodium citrate present in blood is diluted in the systemic circulation and converted to citric acid by reaction with carbonic acid (with generation of bicarbonate).

• Citric acid is then rapidly metabolized to carbon dioxide and water by the Krebs cycle in the mitochondria of tissue cells (predominantly liver and skeletal muscle cells).

• Half-life of citrate in plasma is just 5 minutes (far shorter than that of heparin).

• Anticoagulation with tri-sodium citrate can be associated with disturbance of acid-base balance (metabolic alkalosis), disturbance of blood calcium concentration (hypocalcemia) and disturbance of blood sodium concentration (hypernatremia).

Adverse events

• The most common adverse event related to apheresis is citrate toxicity which may manifest as hypocalcemia on laboratory testing and clinically as paresthesias (numbness in the hands and feet and around the mouth), nausea, vomiting, chills, twitching, tetany, seizures and cardiac arrhythmias. In patients with renal insufficiency, infusion of citrate can also lead to metabolic alkalosis

Apheresis & Anticoagulation

• What is hemostasis

• Components & Stages of hemostasis

• How to measure hemostasis

• Antithrombotic drugs

• Anticoagulants & reversal agents

– Heparin & LMWH

– Warfarin

– DOACs

– Citrate