1. New Trends In The Management Of Bleeding Disorders
Galila Zaher
MRCPath
Consultant Hematologist
KAUH
Major hemorrhage presents a challenging problem to hematologists, anesthetists, surgeons and general physicians.

2. Congenital Bleeding disorders
VWD
Hemophilia A
Hemophilia B
Other congenital factor deficiency
Bernard Solier syndrome
Glanzman’Thrombathenia
Storage pool defect

3. Acquired Bleeding Disorders
Coagulation Factor
Liver Disease
DIC
Consumptions Coagulopathies
Vitamin K deficiency
Platelets defects
ITP
Renal impairment
Myelo-proliferate Disorders

4. Hemophilia A = F VIII deficiency B = F IX deficiency
Affects one in 6000 males
A is 5 X > B
Mild >5,Moderate 2 -5, severe < 2 %
Levels remain stable throughout life
Both HA & HB are X linked
Women are carriers

5. Clinical presentation
< 2 years: joint bleeds
Rare
Only bruising or mouth bleeds are seen
Head injuries are a major concern
> 2 years
Joint and muscle bleeds become more common

6. Indication For Replacement
All joint bleeds: Pain, swelling ,warmth or loss of movement .
Muscle bleeds : severe pain or are in a dangerous location
Bruises usually don’t need treatment
When in doubt

7. Treatment Keep weight off of joint Ice pack
Factor replacement - the sooner the better
Amicar or tranexamic acid : mouth bleed

8. Factor replacement Derived from pooled human plasma
Derived from pig (porcine) plasma
Recombinant products

9. Factor VIII (AHF) Mechanism of action No tool to predict the efficacy
Allergic reactions
Transient (short t ½)
Expensive.
Risk of transmission of infection
FVIII is given IV & activate FX directly
Laboratory Monitoring : no predict the effectiveness of treatment
Contraindications: hypersensitivity, breast-feeding, pregnanc
Adverse reactions: injection or allergic reactions
Plastic syringes :preparation & administration

10. Biotech Development of Recombinant Factors
Human FVII gene
Amplification hF
Gene
Single copy of gene isolated
Liver gene library hF
gene
Activation
and Purification
Expression of rF in culture medium
BHK cells
hF = human factor
BHK = baby hamster kidney

11. Recombinant Factors Advantages : Safe and stable source of the agent
When sources are scarce
Problems :
Contaminating proteins :Infectious or immunogenic agent
Expensive
Sources for product are Scarce making isolation and purification difficult
Contaminating proteinss :Based on the type of biological organism use in the expression process

12. Genetic Study Study the development of inhibitors.
Gene Transfer Sustained therapeutic production of factors with No stimulation of an immune response .
Efforts to bring genetic correction of the more common bleeding disorders such as hemophilia A to the clinic in a safe and accessible manner
Development of inhibitors— how they arise and how to prevent and treat .
Diagnosis and management of VWD, especially in individuals who have borderline levels

13. The Tools of Genetic Engineering
DNA gene fragment of interest
Endonucleases
Plasmid
Ligase
Host that is capable of accepting DNA
Insertion into the genetic machinery
Confirm that the gene is inserted.
Purify the protein of interest
(DNA encoding for protein )
thermally stable “restriction enzymes cut DNA at specific sites
Isolate the plasmid from a bacterial culture and purify it from other contaminating DN

14. Gene Transfer Clinical Trials
5 trials approved in the States .
Retroviral vector :B-domain deletion
Non-viral approach :reduction factor use & spontaneous bleeding episodes.
Gutless adenovirus : eliminate immune response
GENE TRANSFER FOR THE HEMOPHILIAS
To date, 5 different trials, 3 for hemophilia A and 2 for hemophilia B,
40 patients with severe hemophilia.
Given recent scientific and technical developments, genetic correction of hemophilic patients is now viewed as an achievable goal
Many tissues and cell types (skeletal muscle, liver, spleen, and skin) are capable of producing and expressing fully functional FIX protein.
Although there is debate as to the minimally hemostatic factor level, 5% of the normal factor level is sufficient to convert a severe to a moderate or mildly affected level.
Retroviral vector :B-domain deletion : limited integration
Non-viral approach & AAV2 vector : Reduction factor use & spontaneous bleeding episodes.
Gutless adenovirus eliminate the immune response .

15. Results Of Clinical Trials
Long-term therapeutic expression not achieved, but data are encouraging.
Detectable factor levels observed.
The subjective : decreased bleeding .
No evidence of inhibitor.
Hepatic toxicity , thrombocytopenia.
Decline expression .
Genetic correction of the is feasible.
Given the potential immune response to the vector , the development of inhibitors was not detected
Represent a milestone in the use of genetics for treatment of human ailments
Hepatic toxicity (Grade II–III) reported in 2 trials .
Decline expression :gene silencing, immunological clearance, or senescence of the fibroblasts .

16. Shortcoming Of Treatment Modalities
Short T 1/2
Coast
Infections & Immunologic
Hepatic toxicity ,low platelets
Decline expression.
Owing to the shortcoming of treatment Modalities prompted the need for anew hemostatic agent.

17. Initiation of Haemostasis
prothrombin X
VIII/vWF
VIIIa
VIIa TF Xa V Va
thrombin
TF-bearing cell Va XI
XIa TF
VIIa IX
Fibrinogen
platelet
IXa
prothrombin
X X
XIa
Exposed TF as a result of a vessel wall injury TF-FVIIa complexes convert FX into FXa on the TF-bearing cell.
This limited amount of thrombin activates FVIII, FV, FXI and platelets.
Thrombin-activated platelets change shape, resulting in exposure of negatively-charged phospholipids,
which form the perfect template for full thrombin generation involving FVIII and FIX.
In vitro cell models:rFVIIa can bind to activated platelets and generate small amounts of Fxa, independent of the presence of tissue factor.
). It is postulated that the thrombin-generating capacity of rFVIIa is enhanced substantially by platelets This increase in the rate of thrombin formation with large doses of rFVIIa permits the formation of fibrin, which is less susceptible to lysis
. The rFVIIa seems to work in a TF-independent manner directly on factors IX and X on the phospholipid surface of activated platelets. rFVIIa is able to activate factor X on phospholipid vesicles, activated platelets, or monocytes independent of TF, although the TF-independent generation of thrombin is much less efficient than the TF-dependent thrombin generation by rFVIIa.[16
thrombin
Fibrin
IXa
VIIIa Xa Va
activated platelet IX

18. TF–independent mechanism of rFVIIa enhanced hemostasis

19. Rational Thrombin crucial role in haemostasis.
Any agent that enhances the thrombin generation 'general haemostatic agent'.
rFVII enhances thrombin generation on activated platelets
Compensates for lack of FVIII and FIX.
Normalize fibrin clot permeability
any agent that enhances the thrombin generation may be characterised as a 'general haemostatic agent'. ‘
rFVIIa) in high doses (approximately 100 microg/kg) can induce haemostasis in the absence of FVIII and FIX.
The phrase “universal haemostatic agent “was coined by Dr Ulla Hedner when she proposed that rFVIIa might have broad applications in clinical situations characterized by uncontrolled bleeding have broad applications in clinical situations characterized by uncontrolled bleeding

20. Pharmacokinetic t½ :2.7 h Inter-subject variability.
Rapid clearance in children > adults.
No readily available assays
The haemostatic levels remains uncertain.
Frequent bolus injections, IVI potential to minimize usage.
It is a 50 –KD analog of the naturally occurring serine protease
Considerable inter-subject variability.
Plasma FVII:C levels, measured by a one-stage prothombin-based assay, reflect the plasma concentration of rFVIIa:C.
Initial dose µg/Kg Repeat every 2 hours until clinical improvement Increase to q 2-6H if indicated
administration of rFVIIa by continuous infusion has been reported. uncertainty as to whether continuous infusion of rFVIIa has similar therapeutic efficacy to an equivalent total dose administered by bolus injections
Severe FVII deficiency: µg/Kg at 4 –6H intervals shortens/normalizes PT

21. Potential Use Increases thrombin generation on activated platelet
Hemophilia (FVIII/FIX deficiency)
Acquired hemophilia.
Platelet disorders qualitative and quantitative
Diffuse bleeding triggered by surgery and trauma.
Impaired initial hemostasis
FVII-deficiency
Liver disease
Oral anticoagulant therapy
The phrase “universal haemostatic agent “was coined by Dr Ulla Hedner when she proposed that rFVIIa might have broad applications in clinical situations characterized by uncontrolled bleeding
Radical prostatectomy
Valve replacement surgery, cardiac surgery.
Acquired haemophilia spontaneous and surgical bleed in Europe. .
Healthy : uncontrollable bleed following surgery or trauma.
Type III VWD were successfully treated with rFVIIa

22. Hemophilia with inhibitors
FDA Approved Feb 1999
Bleeding during or prior to ITI therapy.
Control bleeding during surgery.
Safe and effective in 92% hemophilia research society registry
Inhibitor titres are not boosted.
Home treatment: mild-moderate episodes.
Recommended dose ug/kg q 2 -6 h or IVI.
Immune tolerance protocols success in 80% .
Safe and effective for bleeding episodes & haemostatic cover for surgical procedures in up to 92%
In conclusion, rFVIIa is the treatment of choice for the management of surgery and acute life- or limb-threatening bleeding in children with haemophilia and high responding inhibitors. Recently, the effectiveness and safety of rFVIIa in patients with hemophilia with inhibitors has been reported in the hemophilia research society registry.[13]

23. Acquired Hemophilia
Rare but potentially life-threatening condition mortality rate 20%.
Auto-antibodies against the deficient factor.
rFVIIa is effective in major bleeding
Induces haemostasis independent of the presence of FVIII or FIX.
Well tolerated in these patients
] However, most are anecdotal case reports and small series. Used successfully in acquired haemophilia.

24. Liver Disease
Reduction in the synthesis of factors involved in coagulation and fibrinolysis.
Moderate thrombocytopenia.
Upper gastrointestinal tract.
Vitamin K .
FFP
PCCs : thromboembolic complications.
that rFVIIa might have broad applications in clinical situations characterized by uncontrolled bleeding. Against this background, it should be no surprise that rFVIIa has attracted such attention across a divers range of medical specialties
Activated PCCs such as FEIBA, Proplex T, and Autoplex T (Baxter Healthcare, Deerfield, IL) have a higher margin of safety with much less risk of viral transmission and a low level of thrombotic events.[20-23]

25. rFVII &Liver Disease
Acute hepatic trauma, liver biopsy, chronic liver disease ,cirrhosis, and liver transplantation.
Experimental studies :seems to be safe and effective.
No evidence of thrombosis .
Cirrhosis , achieved hemostasis in 74%
Jeffers et al
Jeffers et al[33] Patients with cirrhosis (71) evaluated the effect of 4 doses of rFVIIa (5, 20, 80, and 120 µg/kg) on the correction of PT and the time to achieve hemostasis in patients with cirrhosis with coagulopathy who were undergoing laparoscopic liver biopsy.

26. The Risk Of Thrombosis In LIVER Patients
No evidence of dose relationship
Many events have an alternative aetiology
Few events within the first day after dosing
No increase in events as compared with background transplant population

27. Drug-Induced Coagulopathy
Oral anticoagulant treatment  hemorrhage :0.6%/ m .
Vitamin K, FFP or PCCs
rFVIIa in healthy volunteers :50% drop of INR Girard et al
An open, multicenter pilot trial is underway to determine the efficacy
Fondaparinux. normalized PT, aPTT, and TT.
Bijsterveld et al
The incidence of hemorrhage :0.6%/ m at a therapeutic INR.
Bijsterveld et al[50] studied the effect of rFVIIa in neutralizing the anticoagulant effects of subcutaneous fondaparinux. In a randomized, placebo-controlled study of 16 healthy volunteers, rFVIIa normalized the prolonged PT, aPTT, and thrombin time induced by fondaparinux

28. Glanzmann’s Thrombasthenia
Refractory to platelet transfusion
Increases the initial thrombin generation, thereby compensating for defective platelet
Effective in 60% during surgery .
No adverse effects of rFVIIa
International registry data :relatively safe and effective when used in GT.
Blood 1999; 94 (11):
(GT) isautosomal recessive
Glanzmann's thrombasthenia, fibrin – VWF- glycoprotein Ib-V-IX
All-oantibodies to platelet membrane GP IIb/IIIa
rFVIIa alternative to platelet transfusion. might help adhesive hemostasis at low platelet densities by increasing the initial thrombin generation, thereby compensating for the low platelet number
GT)
isautosomal recessive
rFVIIa alternative to platelet transfusion.

29. Reduction in bleeding time in 52.4% of 105 patients .
Thrombocytopenia
Increased thrombin generation on activated platelets compensate for the low platelet number.
Reduction in bleeding time in 52.4% of 105 patients .
Kristensen et al
No major adverse
Severe Thrombocytopenia and life-threatening haemorrhage were successfully managed
These data suggested that rFVIIa might help adhesive hemostasis at low platelet densities by increasing the initial thrombin generation, thereby compensating for the low platelet number.
uremia.
Thromb Haemost 1998; 80:353
Bernard-Soulier syndrome, fibrin by a glycoprotein IIb-IIIa-mediated
enhanced fibrin deposition bypassing the specific defect in thrombocytopathias. Single dose of rFVIIa (90 microg/kg). administration shortened PT, APTT ,BT and clot retraction.

30. Surgical &Trauma patients
Effective and safe in the management of uncontrolled surgical in patients not known to have inherited coagulopathy.
Trauma :surgical intervention failed to stop life- threatening bleeding.
Significant decrease to 2 packed RBC
Shortening of PT & aPTT
Adjunctive hemostatic treatment
Theoretical risk of TED ,none observed
CONCLUSIONS: rFVIIa is found to be a promising adjunctive hemostatic treatment for trauma patients suffering from massive bleeding. Controlled trials are warranted to evaluate the safety and efficacy of this drug

31. Building Strong Scientific Evidence
Clinical area Status on project
Liver transplantation Ph 2 study
Upper GI bleeds Ph 2 study
Liver resection Ph 2 study
BMT Ph 2 study
Reversal of OAC Ph 2 to be started
Traumatology Ph 2 to be started

32. Questions more than answers
Optimal dosage.
Dosing interval.
Adjunctive hemostatic treatment .
‘General haemostatic agent.
Thromboembolic events .
Coat analysis studies.
Need for evidence-based guidelines
Unresolved Issues
Best used alone or in conjunction with platelet transfusion.
. As clinical use and experience increase and the cost of rFVIIa decreases, the indications for the use of rFVIIa may change drastically. The use of rFVIIa is evolving, and the indications, dosage, and precautions or contraindications need to be further described and defined.

33. Local experience Acquired Hemophilia Fresh PR bleeding
FFP. Cryoppt,FVIII conc
In preparation for molar root extract
FVIII conc 100IU/Kg
rFVII 30IU
Normal hemostasis
Tranexamic acid

34. Thank You

35. Amount of thrombin formed in the initial burst is critical to assure
assembly of a thick, strong fibrin plug
activation of FXIII to cross link fibrin
activation of TAFI to make fibrin plug resistant to fibrinolysis

36. RNA repair Pre-messenger RNA (pre-mRNA) repair.
splicing mechanisms to correct a portion of the defective RNA.
The advantage :large genes or genes that contain large regulatory elements.
Injection of a plasmid encoding a pre-mRNA
Useful for the treatment of autosomal dominant disorders.
Future Prospects A novel approach for genetic correction is the use of pre-messenger RNA (pre-mRNA) repair.
A pre-mRNA containing a portion of correct gene sequence is designed to base pair with the pre-mRNA transcribed from the defective gene. The designed pre-mRNA also contains all the requisite splicing signals that allow 2 independent mRNAs to splice together, resulting in a correct copy of mRNA that is translated into a normal protein.

37. Inhibitors &Gene Transfer
Inhibitors :20% HA patients and 3% of HB patients.
Antibodies inactivate the factor by changing conformation.
Depends on type of genetic mutation.
A large deletion  incidence of inhibitor .
Bleeding episodes are difficult to manage
A large gene deletion and complete loss of protein typically lead to a greater incidence of inhibitor
Bleeding episodes in patients with inhibitors are difficult to manage

38. Human/porcine factor VIII
FEIBA
Human/porcine factor VIII
The classical model of the coagulation cascade is to be replaced by a new, cell based model of coagulation emphasizing the interaction of coagulation proteins with cell surfaces of platelets subendothelial cells and the endothelium.