Orthopedic Surgery and Venous Thrombosis: Relationship to Antiphospholipid Antibodies? A Pilot Study Natalia Yazigi MD, Joseph Mazza MD, Hong Liang PhD, Mark Earll MD, William Hocking MD, James Burmester PhD, Steven Yale MD Marshfield Clinic and Marshfield Clinic Research Foundation, Marshfield, Wisconsin

Venothromboembolism: Orthopedic Surgery  Compared to other surgical interventions, patients undergoing major orthopedic joint reconstructive procedures have one of the highest rates of thromboembolic disease.  Without adequate mechanical or anticoagulation prophylaxis, 50% to 80% of total hip and knee arthoplasties will develop deep venous thrombosis (DVT).

Venothromboembolism: Orthopedic Surgery (cont’d)  Total pulmonary embolism (PE) rates for hip arthroplasty ranges between 0.9 to 28% (fatal PE %); total PE rates for knee arthroplasty ranges between 1.5 to 10 (fatal PE %).  With pre-and post-operative anticoagulation regimens commonly used in these patients, the incidence of DVT has been reduced to 12-15%.

Incidence of VTE Within 91 Days of Surgery Among Patients Without a Malignancy Surgical procedure% total95% CI Total hip arthroplasty Total knee arthroplasty ORIF femur Shoulder arthroplasty Thyroid surgery Open cholecystectomy Nephrectomy Total abdominal hysterectomy Replacement of the heart valve White R et al, Thromb Heamost 2003, 90:446

Incidence DVT in Total Hip Arthroplasty He Xing K. et al, Thrombosis Research, 2008, 123:24–34

Thrombotic Stimuli During Total Hip and Knee Arthoplasty  Venous stasis:  Venous stasis: Tourniquet placement, immobility during the postoperative period  Endothelial injury:  Endothelial injury: Manipulation during preparation of the femoral prosthesis releases tissue thromboplastin and other thrombogenic molecules  Hypercoaguability:  Hypercoaguability: Thrombogenic stimuli - reduction in antithrombin III and inhibition of fibrinolysis due to blood loss

Unexplained Factors for VTE in Orthopedic Surgery  Hypothesis Bone contains a large concentration of phospholipids, which are found as structural components of fat and hematopoietic cells that comprise the bone marrow (hidden epitopes). Disruption or invasion of this site as a consequence of surgery causes inflammation and cellular apoptosis leading to turnover of phospholipid membranes.

Unexplained Factors for VTE in Orthopedic Surgery  Exposure of phospholipids, to normal immune surveillance, thereby eliciting an immunological response.  Production of autoantibodies targeting phospholipid binding proteins, prothrombin complex, and membrane phospholipids (essential components of the coagulation cascade).

Antiphospholipid Antibodies (APLa)  Diverse group of autoantibodies that bind to a variety of antigens including: (1)phospholipid binding proteins: 2GP1, prothrombin, protein c, protein s, annexin CV, HMW kininogen, complement factor H, and factor XI, which are components expressed or bound to the surface of vascular endothelial cells, platelets or other cells (2) negatively charged phospholipids (3) phospholipid-protein complexes

Hypercoagulability in APLa: Proposed Mechanism  Proposed mechanisms for the prothrombotic state induced by APLa include: interference with prothrombin conversion to thrombin inhibition of activation of protein C and S pathways activation of platelets, monocytes and endothelial cells promotion of tissue factor synthesis and expression by suppression of the inhibitory activity of tissue factor pathway inhibitor (TFPI) interference with fibronolysis by inhibiting the conversion of plasminogen to plasmin

Procoagulant and Anticoagulant Phospholipid-bound Protein Targets for Antiphospholipid Antibodies (APLa)

What are APLa’s?  Include among others, Lupus anticoagulants, B-2 Glycoprotein 1 and anticardiolipin antibodies.  Immunoglobulins that react with heterogeneous epitopes on β2-GPI or prothrombin, and result in tests positive for lupus anticoagulant activity (coagulation assays) or anticardiolipin antibody.  Due to the heterogeneity of these antibodies some antibodies may exhibit lupus anticoagulant effects while others are only active in anticardiolipin assays.  Thus APLa are closely related, overlapping but not identical antibodies.

APLa Frequency and Significance  APLa can be found in the serum of individuals of all ages, in patients who are entirely asymptomatic and without a history of VTE. IgG ACLa have been found in:  12 to 52% of clinically healthy elderly persons  2% of a younger population  1 to 9% of blood donors  The presence of these antibodies in apparently healthy individuals has led to speculation that they have a physiologic function that remains to be elucidated.

APLa Detection  Screening direct activated partial thromboplastin time (dAPTT) prothrombin time (dPT) kaolin clotting time dilute Russel Viper Venom test (dRVVT)  Because Russel Viper Venom (RVVT) directly activates coagulation factor X, it is considered the most sensitive laboratory assay for LA

What are APLa’s?  Neither screening test (ACLa or LA), is a 100% sensitive  In general, ACL test are more sensitive wheras 2 GP1 and LA have a higher degree of specificity for APS  LA, medium to high titers of IgG ACL and IgG 2 GP1 antibodies are most strongly associated with thrombosis

APLa Diagnosis  The diagnosis of APS can be made only when a characteristic clinical presentation (vascular thrombosis, pregnancy morbidity) is combined with objective laboratory abnormalities (lupus anticoagulant, anticardiolipin Ab, Antiβ2-GP1 Ab) are present on 2 or more occasions at least 12 weeks apart.

What is Lupus Anticoagulant?  The LA phenomenon reflects the in vitro inhibition by antiphospholipid antibodies (anti-2GP1 and anti- prothrombin antibodies) of phospholipid- dependent coagulation reactions.  LA is a laboratory assay that tests for the presence of antibodies (APLa) that competes with coagulation proteins normally found on phospholipid templates thereby inhibiting the conversion of prothrombin to thrombin.  By preventing the formation of the prothrombinase complex (Xa, Va and prothrombin) and subsequent fibrin clot, APLa interfere with phospholipid dependent coagulation pathways as reflected by the prolonged APTT and dilute Russel Venom Time (dRVVT).

What are 2 GP1 APLa’s?  2 GP1 antibodies are useful to confirm the diagnosis in patients with low positive IgG ACL or only IgM or IgA ACLa positivity, in patients with unusual or equivocal features, or in those with negative ACLa and lupus anticoagulant in the presence of highly suggestive clinical features.  2 GP1 antibodies have been identified in up to 10% of patients testing negative for ACL and lupus anticoagulant and clinical features of APS.

Anticardiolipin APLa  ACL (IgG, IgM, or IgA) antibody APLa found in some patients with APS  ACLa antibodies are measured using solid phase immunoassay using 2 GP1 cofactor bound to negatively charged phospholipid, cardiolipin, or other anionic phospholipids Unlike LA, the detection of ACLa is not effected by anticoagulation therapy

Pilot Study Overview  Explore whether activation of the immune response as a consequence of de-novo auto-antigen exposure to phospholipids released during orthopedic (hip or knee replacement) surgery contributes to hypercoaguable states.

Objectives  Goal To determine whether elevated titers of Antiphospholipid antibodies (APLa) occurring as a result of tissue damage in the operative period correlates with the development and incidence of VTE.

Specific Goals  Primary goal Estimate the prevalence of elevated APLa titers that develop after postoperative hip and knee replacement surgery.  Secondary goal Evaluate the association between the presence of elevated APLa titers and the occurrence of symptomatic venothromboembolic events.

Study Design  Prospective cohort study design  150 patients undergoing total knee or hip arthroplasty  The trial will consist of up to a 30 day screening period  Day 1 is designated as the day of surgery  Laboratory testing for APLa will occur on the day prior to surgery, and Days 7 ±2, 14 ± 2, and 21 ± 2 postoperatively  Subjects will be contacted by telephone on Day 56 ±2 to determine whether a venothromboembolic (DVT or pulmonary embolism) event (VTE) had occurred. If a VTE was present, information will be obtained regarding the location and site of thrombosis and method of diagnosis.

Study Methods  Data will be obtained regarding previous history or VTE and whether patients have medical conditions, known to increase the risk of VTE, within 30 days prior to preoperative baseline blood draw.  Primary outcomes will include determination of titers of ACLa and β2GP1 antibodies and clotting time anomalies as detected by lupus anticoagulant and incidence of thromboembolism.  The results of the doppler duplex ultrasound, ventilation/perfusion scan, and/or chest spiral computed tomography will be recorded and the occurrence of postoperative DVT or pulmonary embolism will be correlated with the results of preoperative and postoperative APLa levels.

Inclusion Criteria  Male or female ≥ 18 years of age who are scheduled for primary elective unilateral total knee or hip arthroplasty (i.e. first time the knee or hip is being replaced on the operative side).

Exclusion Criteria  Subjects with a history of objectively diagnosed deep venous thrombosis or pulmonary embolism  Current anticoagulation therapy  Positive APLa (ACLa or β2GPI) antibodies or LA test previously or at the time of preoperative assessment  Known autoimmune disorder such as scleroderma, systemic lupus erythematosus and rheumatoid arthritis  Subjects with an active malignancy  Subjects with an active infection  Resident of a nursing home or long-term care facility  Subjects who will be inaccessible due to geographic or social factors during treatment or follow-up

Results: Positive Distribution PreDay 7Day 14Day 21Total LA0% (0/52) 71.15% (37/52) 71.15% (37/52) 75.00% (39/52) 78.82% (41/52) ACLa0% (0/52) 1.92% (1/52) 1.92% (1/52) 1.92% (1/52) 1.92% (1/52) GP10% (0/52) 1.92% (1/52) 1.92% (1/52) 1.92% (1/52) 3.85% (2/52) Total0% (0/52) 71.15% (37/52) 71.15% (37/52) 75.00% (39/52) The Table above showed that the rate of LA positive is relatively high (71.15%, 71.15%, and 75.00% on day 7, day 14 and day 21, respectively), while the rates of ACLa and GP1 positive are low (less than 2% on day 7, day 14 and day 21).