1. 2Haemophilia Department, Royal Free Hospital, London, United Kingdom
Bleeding diathesis, thrombotic tendencies and endothelial dysfunction in systemic AL patients
1Division of Medicine, Royal Free Hospital Campus, National Amyloidosis Centre, University College of London Medical School, London, United Kingdom
2Haemophilia Department, Royal Free Hospital, London, United Kingdom
Dr Shameem Mahmood
November 2015

2. Background
Systemic light chain (AL) amyloidosis is known to be associated with an increased bleeding diathesis, including the pathognomonic peri-orbital bruising.
Little data exists as to the interplay of different coagulation factors, and prothrombotic risk in these patients, where thromboprophylaxis is an inherent part of our day to day practice.
Von Willebrand Factor (vWF) is a multimeric adhesive glycoprotein which promotes platelet adhesion to the subendothelium at sites of vascular injury and platelet-platelet interactions under high shear- rate conditions. VWF acts as a carrier for FVIII, acting to protect FVIII from rapid proteolysis. It has also been used as a surrogate marker in endothelial dysfunction.
Immunoglobulin light chains form amyloid fibrils and are deposited in different tissues in systemic light chain (AL) amyloidosis.

3. Aims
We sought to report the coagulation abnormalities in newly diagnosed systemic AL patients:
Bleeding risks
Coagulation abnormalities and any prognostic implications
Endothelial dysfunction
Thrombotic risks in patients with a low albumin level

4. Methods
We conducted a prospective study on 100 patients with suspected light chain amyloidosis, with 74 systemic AL between May-December 2013.
Each patient had a detailed baseline assessment of organ involvement as per standard protocol, and completed a Royal Free Hospital v4 adapted bleeding questionnaire.
Assays for vWF:Ag, Protein S, Protein C, Anti-thrombin III, fibrinogen, ADAMTS13, and all clotting factors were performed.
We assessed the endothelial dysfunction using vWF antigen as a surrogate marker following chemotherapy.

5. Results – bleeding risks
22 patients reported bleeding symptoms by the bleeding questionnaire, cutaneous (n=17), oral (n=7), epistaxis (n=5), haemarthrosis (n=1), muscle haematoma (n=1) and following surgery (n=2).
Symptom duration – 4 (0.5-36) months
Factor X deficiency – 2(2.7%)
Prolongation of the PT, APTT and TT occurring in 5 (6.7%), 5 (6.7%) and 35 (47.3%).
Elevated fibrinogen levels were present in 42 (56.8%)
Analysis included 74 patients with systemic AL, 29 (39%) male and 32 (43%) with cardiac involvement, Mayo stage 3 in 29 (39%).
Elevated fibrinogen levels were found in 42 (56.8%), FVIII 67 (90.5%) and vWF:Ag 67 (90.5%).

6. Baseline patient characteristics

7. Results – unexpected findings
Scatter plots comparing FVIII and vWF:Ag levels in all
patient groups respectively.
Von Willebrand factor has a dual role in haemostasis: firstly promoting platelet adhesion to thrombogeneic surfaces and ensuring platelet-platelet cohesion during thrombus formation, and secondly to act as a carrier for FVIII; important in its production, stabilisation, conformation and immunogenicity

8. Overall survival stratified
according to vWF:Ag and
FVIII>280IU/L respectively.

9. Variables associated with survival
Separate multivariate models were performed analysing FVIII and vWF Ag individually due to the close correlation and co-dependant relationship. This analysis showed FVIII> 280IU/dL, log NT-proBNP and a dFLC>180mg/L remained significant, with vWF Ag non-significantly linked with survival.

10. Results – Endothelial dysfunction Pre and post chemotherapy
comparison of vWF:Ag and FVIII
It is difficult to examine endothelial light chain toxicity. Patients re-attending for their follow up visit 6-12 months (n=22 ) following chemotherapy had blood samples taken for vWF:Ag, FVIII and fibrinogen analysis. It was not feasible to arrange follow up blood samples in 26 patients due to their appointment arrangement with us in the time frame of this study and 26 patients died. 19 patients exhibited a fall in the vWF:Ag, (figure 3A) with a median pre and post chemotherapy 360IU/dL and 277IU/dL respectively. Fewer patients (n=4) hadving a corresponding fall in the FVIII level, with pre and post chemotherapy levels 272 IU/dL and 277IU/dL.
Various experiments have attempted to explore the underlying mechanisms of light chain toxicity, with no correlative factor for endothelial dysfunction. Previous studies have proposed vWF as a marker of endothelial damage or dysfunction; which is secondary to a pro-inflammatory and pro-coagulant state. The increased vWF:Ag and vWF:CB are likely to result in increased platelet plug formation. Bauer et al described the concept of vWF fibres laid down within the lumen of the cancer associated vessel wall, with tumour derived VEGF inducing angiogenesis, thus affecting the pathophysiology and activation of the endothelium.{Borsig, 2015 #6192} In systemic AL amyloidosis, light chains misfold and aggregate as amyloid fibrils in vital organs.

11. Results – Thrombotic risk with an albumin less than 25g/L.
Systemic AL - an albumin less than 25g/L, significantly correlated with an elevated vWF:Ag, FVIII, FV and fibrinogen, and reduced levels of protein S and anti-thrombin.

12. Discussion/Conclusion
Newly diagnosed systemic AL patients are at risk of increased pro-thrombotic and bleeding diathesis.
There is no questionnaire which reflects the degree of bleeding risk, with cutaneous vessel fragility accounting for most. FX deficiency was present in 2 (2.7%).
One striking finding was an elevated vWF:Ag and FVIII in greater than 90% of newly diagnosed systemic AL patients, which may reflect a higher prothrombotic environment and likely endothelial dysfunction.
An elevated FVIII and vWF:Ag level greater than 280IU/L carried a significant survival disadvantage. The reasons are unclear, but may arise from endothelial damage and thromboembolic consequences.
We also examined the effects of chemotherapy in a subset of patients, showing that a fall in vWF:Ag levels may reflect endothelial changes.

13. Discussion/Conclusion
Understanding of the coagulation cascade has shown that elevated levels of FVIII and fibrinogen, lower levels of protein S and anti-thrombin contribute to a prothrombotic tendency. The latter findings were found in patients with an albumin less than 25g/L. This raises the issue of strong consideration for anticoagulation in these patients.
Systemic AL carries bleeding risks, thrombotic risks with the vascular endothelium an important factor in this equation.
Our study raises the hypothesis of the extent of endothelial dysfunction as a poor outcome predictor of survival, with changes in this environment contributing to the light chain toxicity. Elevated factor VIII levels have previously been examined in the thrombotic setting, and may reflect microvascular thromboembolism and hence negatively impacting survival.
Recently further exploration to examine the dysregulation of an autophagic flux in the setting of proteotoxicity of the light chain has shown to be important in those with cardiac AL,{Guan, 2014 #6196} Guan et al found that inhibition of the autophagic flux, specifically lysosomal dysfunction is important in the AL-light chain cardiotoxicity and hence development of AL cardiomyopathy with in vitro experiments showing lysosomal dysfunction is an early point in the cascade of events that occur in cardiac AL, specifically the light chain toxicity, followed by mitochondrial dysfunction, ROS dysfunction and consequent cell death and dysfunction

14. Thank you Risk Factors Endothelial Dysfunction Bleeding Thrombosis
Acknowledgements
Patients
Ashutosh D Wechalekar
Sajitha Sachchithanantham
Thirusha Lane
Darren Foard
Taryn Youngstein
Rabya Sayed
Ketna Patel
Marianna Fontana
Candida Quarta
Carol J Whelan
Helen J Lachmann
Julian D Gillmore
Philip N Hawkins
National Amyloidosis Centre clinical staff
Endothelial
Dysfunction
Bleeding
Thrombosis
Treatment
Haemophilia Department,
Royal Free Hospital
Anne Riddle
Keith Gomez