Components of Hemostasis Primary Hemostasis (Platelets &vWF)

Coagulation Cascade (Intrinsic & Extrinsic) Inhibitors of Coagulation (Natural & Acquired) Fibrinolytic System (Activators, Inhibitors &Degradation products)

Activated partial thromboplastin time (aPTT)Derives its name from use of a partial activated Component like the phospholipid cephalin and Negatively charged substance like kaolin for activation Expressed in seconds with normal range (27-40)

Prolonged:Heparin therapy Presence of anticoagulant (lupus like) Factor deficiency Massive blood transfusion. Liver disease High dose coumadin anticoagulation

Factor InhibitorsThe mixing study The patient's serum is mixed with normal serum and the aPTT of this mixture is measured. A 50:50 mixture will correct a factor deficiency (only 30% activity is needed for a normal aPTT). In the presence of an inhibitor, a 50:50 mix will not correct the abnormal coagulation test. If inhibition found, additional tests with diluted patient serum and normal serum will be applied and level of inhibitor will be determined (Bethesda units) by the dilution activity Factor VIII inhibitors IgG mostly, Occur primarily in haemophilia A patients who have received blood component transfusions. They develop a severe coagulopathy that is very difficult to manage May occur in a variety of unrelated conditions with a coagulopathy that is variable and usually disappears spontaneously.

Inhibitors of coagulation and fibrinolysis

Half life (hrs) Protein C Protein S Thrombo modulin 4 42 ND

Chromosome 2q13-14 3p11.1-11.2 20p11.2-cen

Function Protease (FV&FVIII) APC-cofactor Receptor for Thrombin/Prot.C Receptor for prot.C/APC Protease inhibitor Protease inhibitor Protease inhibitor

Protein C receptor Antithrombin TFPI Heparin Cofactor II

ND 70 ND 60

20q11.2 1q23-25 2q31-32.1 22q11

Activation by thrombin or FXa APC inactivates FVa FV-Leiden has Gln instead of Arg at 506, which confer APCR

sEPCR

Protein C pathwayAPCFVIIIa

sEPCR

PC T T TM PCEPCR EPCRsEPCR

FVIIIa

FVa APC FVa S

History 1965 Antithrombin (Egeberg) 1965 dysfibrinogenemia (Beck) 1981 - Protein C (Griffin) 1984 - Protein S (Comp) 1993- APCR (Dahlback) 1994 Factor V Leiden (Bertina) 1996 - PT G20210A mutation (Poort)

Large vessel - Death Medium vessel - Respiratory Failure Small vessel- asymptomatic repetition pulmonary hypertension

Block

Embolus

Lysed / Organized

Extend

Thrombus

40 35 30

of % VTE among autopsie s

25 20 15 10 5 035% 26 % 9 .4%

VTE PE Fatal PE Accountsfor about 10% of deaths in hospitalised patients11.Lindblad B, et al. BMJ 1991;302:70911 2. Dahl OE, Bergqvist D. Curr Opin Pulm Med 2002;8:3947

Venous Thrombosis Makes News in Washington Surgeon General nominated VTE Task Force

: Inherited Common- )Factor V G1691A )Leiden Prothrombin G20210A Increased levels of FVIII and Fibrinogen RareAntithrombin deficiency Protein C deficiency Protein S deficiency Very rare- Dysfibrinogenemia Homozygous homocystinuria ? Future risk factors

:AcquiredTrauma or surgery Immobilization Increasing age Malignant disorders Myeloproliferative disorders Previous thrombosis Pregnancy and puerperium Use of contraceptives or HRT Activated protein C resistance Mild to moderate hyperhomocystinemia Antiphospholipid syndrome

.. 64 . ( )DVT . , . : APS -01 , , - . 5 , , MRI .B.E - Laboratory findings )83-72 .71 (C )23 .54 (C )51 < 34 (C )3.1 < 2.9 (C 8.1 )52.1 < 2.14 (C ) 2 > 1.45 (C normal 75u/ml )51 < (C )002 < 417 u/ml (C )05 < 190 u/ml (C

Coagulation aPTT aPTT mixed with plasma CAI RVVT RVVT confirm TTI APCR Factor V Leiden Immunology anticardiolipin .anti-nuclear ab anti-DNA

Endothelial

cell mediated: injury to EC, receptor induction, increased TF expression, induction of apoptosis Protein C pathway related: aPL binds PC&S inhibition of PC activation, acquired APCR Inhibition of heparin-AT complexes Cross reaction with OX-LDL Increase PAI-1 Platelet activation

Clinical criteria

1.Vascular thrombosis )one or more ATE or VTE) 2. Pregnancy morbidity a. one or more IUFD >10th week b. one or more premature birth, preeclampsia, placental insufficiency, abruption, IUGR c. 3 or more early )10th week) abortions,) >2 late)

Laboratory criteria

1. Anti CL Ab )IgG/M), on two )6w) occasions 2. LAC on two )6w) occasions )aPTT, DRVVT) 3. Exclusion of other coagulopathies Definite aPLS is establish by at least one criterion of each category

Yield (%) 80 70 60 50 40 30 20 10 0

Selected Unselected until 1993 from 1993

Thrombophilia

Healthy subjects N affected % 0.2-0.4

Unselected patients N 2,028 2,028 affected % 3.7 2.3 1.9 18.8

Selected patients N 880 752 752 162 affected % 5.2 7.4 4.6 40

Protein C Protein S Antithrombin

15,070 ND 9,669

0.02 4.8 0.05 2.7 0.06

2,028 1,142

Factor V Leiden 16,1502,192

PT G20210A

11,932 1,811

2,884

7.1

551

16

Thrombophilia

Healthy subjects N affected %

Unselected patients N affected %

Selected patients N % affected

FVIII APCR Hcy

534 445 1,153

11.8 8.1 6.1

534 337 856

23.2 23.4 11.7

60

56.7

Priority for testing HighAPCR Factor V Leiden PT G20210A Homocysteine Factor VIII Lupus anticoagulant

IntermediateProtein C activity

LowFibrinogen

Free protein S antigen Thrombin time Antithrombin activity FIX activity Anticardiolipin Abs FXI activity C677T MTHFR

Risk of recurrence of thrombosis according to type of thrombophiliaHigh Antithrombin APL Abs Intermediate Increased Protein C Protein S FVIII Hcy None FVL FII

Prothrombin F1+2 and soluble fibrin in normal pregnancy

S Sarig )Fertility Sterility 2002(

Gris ( Thromb Haemost )1999

74, . , 81 ", . . , . , . , .

13

A) Cabin related Cramped sitting position More in economy class 83% Non-aisle seats Lower air pressure, relative hypoxia differ among airlines Low humidity and dehydration

B) Passenger related Age over 40 Previous VTE Thrombophilia Hormonal Therapy Pregnancy Varicose veins Cancer Overweight

Coagulation activation in hypobaric chamber

Armand Trousseau (1801-1867)

When you are undecided about the nature of the disease of the stomach, when you hesitate among a chronic gastritis, a simple ulcer, and a carcinoma, a phlegmatia alba dolens (thrombophlebitis) occurring in the leg or arm will put an end to your indecision and you will be able to assert positivelythat a cancer is present.

Endothelial damage Shift to procoagulant endothelium Invasion of cancer cells into vessel wall

Stasis of blood Frequent immobilization, surgery Compression of blood vessels by tumor

Changes in the blood constituents Activation of clotting proteins and blood

cells

Tumor Cell Hemostatic Properties and Tumor BiologyTumor cell hemostatic properties Procoagulant ActivitiesTissue Factor

Mechanisms of malignancy

Fibrinolytic Activities(t-PA, u-PA, u-PAR, PAI)

Coagulationdependent

PROLIFERATION ANGIOGENESIS

TC-derived Cytokines(IL-1, TNF, VEGF)

Coagulationindependent

METASTASIS

Cell Adhesion Molecules

Tissue Factor/FV IIa Factor Xa Thrombin

Growth Invasion Metastasis

Fibrin generation plays additional roles in Angiogene these processes

Tumor cell TF FVIIa

FXProthrombin

FXaThrombin

Fibrinogen

VEGF Angiogenesis TF

FIBRIN

IL-8

Endothelial cellsRickles FR, and Falanga A. Thromb Res 2001

Tumour Cell- Host Cell Interactions at The Vascular Sitefibrin Localized clotting activation P PInduction of monocyte procoagulant activity

Induction of endothelial procoagulant and adhesive properties

TCP P

TC

TC

P P P

PMN fibrin P TC

Endothelial Cells

TC = tumor cell P = platelet PMN = polymorphonuclear leukocyte

Prandoni, Falanga, Piccioli, Lancet Oncology, 2005

The

bleeding diathesis of APL is most consistent with the diagnosis of DIC. Primary fibrinolysis is hard to document in APL and probably does not exist. Excessive fibrinolysis is probably secondary to thrombin generation. ATRA can improve the coagulopathy in patients with APL.

sEPCR

Protein C pathwayAPCFVIIIa

sEPCR

PC T T TM PCEPCR EPCRsEPCR

FVIIIa

FVa APC FVa S

Acquired activated protein C resistance is common in cancer patients and is associated with VTECancer with VTE Cancer without VTE VTE without Cancer Normal Controls

Patients FV Leiden APCR without FV Leiden

55 1 (2%) 29 (54%)

58 4 (7%) 9 (17%)

54 18 (33%) 7 (19%)

56 2 (4%) 0

Haim, Am J Med 2001

:HeparanaseHeparanase activity was implicated in cellular invasion ,angiogenesis, inflammation and cancer metastasis

Heparanase up-regulation was documented in an increasing number of primary solid and hematological .human malignancies Heparanase up-regulation correlated with reduced postoperative survival of pancreatic, bladder, gastric, .cervical and colorectal cancer patients Similarly, heparanase up-regulation correlated with increased lymph node and distant metastases,

Hepa

Hepa Hepa

Heparan sulfate

Considerations in planning treatment of patients with thrombosis Efficacy of treatment Rate of bleeding complications Rate of recurrence Complications due to recurrence

PT

correlates initially with FVII thus not reflecting actual AC Protein C fast drop creates initial hypercoagulability Initiation of coumadin should be covered by heparin

INR - International Normalized RatioISI

INR = R R = PT (sec) patient / PT control example: PT coumadin treated patient - 32 sec, PT control - 12 sec R = 2.6ISI = international standartization index

Intrinsic pathway Antithrombin ATIII ATIII ATII I

Extrinsic pathway

Xa

Xa

Pentasaccharide

IIFibrinogen

IIaFibrin clot

Adapted with permission from Turpie et al . N Engl J Med.2001;344:619

ORALTTP889 X TF/VIIa IX IXa VIIIa Rivaroxab an APIXABAN YM150 DU-176b Ximelagatran Dabigatran Fibrinogen Va Xa II AT

PARENTERALTFPI )tifacogin)

)APC (drotrecogin alfa )sTM )ART-123 Fondaparinux Idraparinux

DX-9065a IIa Fibrin

Adapted from Weitz & Bates, J Thromb Haemost2005