Procoagulant Genetic Factors in a Pooled Cohort of 582 Chronic Fatigue Syndrome, Fibromyalgia and Related Chronic Illness Cases1. H. Harrison1,2, C. A. Ryser3, J. Brewer4,5, and D. Berg2. Univ AZ Col of Med1, and HEMEX Labs2, Phoenix, AZ; Health Centers of America3, Plaza Internal Med4, and Univ MO School of Med5, Kansas City, MO. Objectives: Hypercoagulability due to procoagulant (thrombophilic or hypofibrinolytic) genetic factors is commonly evaluated in the context of thromboembolic disease, cardiac, and/or gynecologic/obstetric risks. We have reported low level activation of coagulation in patients with active Chronic Fatigue Syndrome and/or Fibromyalgia (CFS/FM) [Ref: Blood Coag Fibrinol 10:435-438] and have also found an association of CFS with increased defects in thrombophilia and hypofibrinolytic activities in a cohort of patients with HHV6 and CFS. We postulate that such procoagulant genetic defects may be predispositional or permissive in individuals who become chronically ill. These studies were done in well-defined, yet limited cohorts that had sample sizes too small to statistically evaluate whether there was a significant additive or polygenic contribution if more than one marker was positive, and too small to detect enrichment of the less prevalent genetic deficiencies. This study represents the results of “database mining” to achieve a larger N. Methods: We analyzed a database of 582 adults screened for suspected CFS/FM or related chronic illness and whose testing included both the ISAC and HTRP panels of coagulation testing. We used the ISAC panel of tests (fibrinogen, prothrombin fragment 1+2, thrombin-antithrombin complexes, soluble fibrin monomer, and platelet surface P-selectin) for measuring low-level activation of coagulation. For Hereditary Thrombotic Risk Panel (HTRP) data, we analyzed seven markers with known Mendelian characteristics: antithrombin, protein C, protein S, activated protein C resistance (APCR), prothrombin (F2), lipoprotein Lp(a), and plasminogen activator inhibitor-1 (PAI-1). Results: The cohort was stratified by ISAC scores (one point for each abnormal result) and HTRP scores (one point for each positive genetic factor). 72% (421/582) of patients had positive ISAC results as defined by the criterion of two or more results being abnormal; 69% (404/582) were positive for one or more genetic markers. All marker abnormality rates were increased over general population values. The HTRP abnormality rate increased as a proportionate function of ISAC score up to 81% HTRP-positivity with 4 ISAC results positive. Stepwise increments in abnormality rates were evident for APCR (to 0.155), F2 levels (to 0.512), and PAI-1 levels (to 0.241). Lipoprotein Lp(a), a hypofibrinolytic protein, was increased in more than 30% of all patients regardless of ISAC score. Overall 69.4% of the cohort was positive for one or more of the procoagulant protein markers [gp=20%], 25.4% for two or more [gp=4%], and 4.5% for 3 or more [gp=0.8%]. Analysis of the positive predictive value and likelihood ratios of HTRP score and ISAC-positivity shows that a patient with an HTRP score of 2 or more is 3.5 times more likely to be ISAC-positive than one with an HTRP score of 1 or less. Conclusions: These data support the general hypothesis of concerted genetic contribution(s) of coagulation protein abnormalities to CFS/FM and are consistent with family histories. The pooled CFS/FM and related chronic illnesses are associated with increased rates of abnormalities for all markers except APCR. CFS patients with normal HTRP results may warrant testing for rarer inheritable thrombopathies. Genetic testing to identify the type of procoagulant defect may be warranted as effective interventions to prevent or treat CFS/FM become available. 1) Oral Presentation, 7th International Biennial Conference on Chronic Fatigue Syndrome, Oct, 2004, Madison, WI, USA – In Press

Procoagulant Genetic Factors in a Pooled Procoagulant Genetic Factors in a Pooled Cohort of 582 Chronic Fatigue Syndrome, Cohort of 582 Chronic Fatigue Syndrome,

Fibromyalgia and Related Chronic Fibromyalgia and Related Chronic Illnesses Illnesses 11

Harold H. Harrison, M.D., Ph.D.*Harold H. Harrison, M.D., Ph.D.*,1,2,,1,2,, Carol Ann , Carol Ann RyserRyser, , M.D.M.D.33, Joseph H. Brewer, M.D. , Joseph H. Brewer, M.D. 4,54,5, David E. Berg, M.S , David E. Berg, M.S 22..

University of Arizona College of Medicine University of Arizona College of Medicine 11 and HEMEX and HEMEX Laboratories Laboratories 22, Phoenix, AZ; Health Centers of America , Phoenix, AZ; Health Centers of America 33, Plaza , Plaza

Internal Medicine Internal Medicine 44 and University of Missouri School of Medicineand University of Missouri School of Medicine55, , Kansas City, MO.Kansas City, MO.

1) AACFS Lecture, Oct, 2004, Madison, WI: In Press*E-mail: Genedoc169@aol.com

CHRONIC FATIGUE SYNDROMECHRONIC FATIGUE SYNDROMECoagulation Testing in CFSCoagulation Testing in CFS

New findings indicate that a significant proportion of patientswith CFS/FM have a low level, subthrombotic activation of coagulation via immune-mediated APS-like pathophysiology.(Ref: Blood Coag Fibrinol 10: 435-8, 1999.)

This Immune System Activation of Coagulation (ISAC) is detectedby measuring five assays of plasma proteins and platelet function:

1) Fibrinogen (increased)2) Prothrombin cleavage fragment 1+2 (F1+2, increased)3) Thrombin / antithrombin complexes (TAT, increased)4) Soluble fibrin monomer (SFM, increased)5) Platelet activation (surface CD62P, increased)

Low Level Activation of Coagulation in Low Level Activation of Coagulation in Chronic Fatigue Syndrome and FibromyalgiaChronic Fatigue Syndrome and Fibromyalgia

Initial Study: CaseInitial Study: Case--Control, AssociativeControl, Associative

1. Patients (54), Controls (23).

2. Patients assigned as CFS by CDC/NIH criteria.

3. Healthy controls matched for age, sex, sample travel.

4. Three clinical sites (Pt / Ctl), N:Rochester, NY (15 pts / 5 ctls, N=20)Houston, TX (19 pts, 8 ctls, N=27)Phoenix, AZ (20 pts, 10 ctls, N=30)

Ref: Blood Coag Fibrinol 10: 435-8, 1999.

Low Level Activation of Coagulation in Low Level Activation of Coagulation in Chronic Fatigue Syndrome and FibromyalgiaChronic Fatigue Syndrome and Fibromyalgia

Table 1. ISAC* test results for CFS/FM patients and controlsTable 1. ISAC* test results for CFS/FM patients and controlsTest FIB F1+2 TAT SFM CD62P PA Score 2 or more

Ref Range 180-310 mg/dL 0 - 1.1 nmol/L 1.0 - 4.1 ug/L 0 - 17 nmol/L 0 - 27% Normal None

A. Controls (N=23)(#Abn / N) 2 / 23 3 / 23 4 / 23 3 / 23 5 / 23 0 / 23 1 / 23

rate 0.087 0.130 0.174 0.130 0.217 0.000 0.043Mean 280 1.0 1.6 10 17.5

B. Patients (N=54)(#Abn / N) 45 / 54 26 / 54 25 / 54 32 / 54 21 / 54 22 / 54 50 / 54

rate 0.833 0.481 0.463 0.593 0.389 0.407 0.926Mean 367 1.2 1.6 22 22

P Value <0.001 <0.005 <0.005 <0.001 <0.10 <0.001 <0.001

*ISAC (Immune System Activation of Coagulation) panel includes: fibrinogen (FIB), prothrombin cleavage fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), soluble fibrin monomer (SFM), and platelet surface P-selectin (CD62P) before and after stimulation by ADP (PA score).

Ref: Blood Coag Fibrinol 10: 435-8, 1999.

Low Level Activation of Coagulation in Low Level Activation of Coagulation in Chronic Fatigue Syndrome and FibromyalgiaChronic Fatigue Syndrome and Fibromyalgia

ConclusionsConclusions1. Majority of patients (92.6%) with clinically-defined CFS/FM

have a low level activation of coagulation.

2. Strongest discriminant for separating patients from controls is “two or more” of 5 test results positive in the ISAC panel.

3. May be a final common pathway of pathogen induction of antiphospholipid antibodies and endothelial activation.

4. Demonstrates organic basis of illness and potential utility of anticoagulation treatment.

5. Hereditary influences of thrombophilia or hypofibrinolysis may predispose or be permissive to activation in CFS/FM.

Ref: Blood Coag Fibrinol 10: 435-8, 1999.

Low Level Activation of Coagulation in Low Level Activation of Coagulation in Chronic Fatigue Syndrome and FibromyalgiaChronic Fatigue Syndrome and Fibromyalgia

Genetic Influence HypothesisGenetic Influence Hypothesis

If procoagulant genetic factors, either thrombophilic or hypofibrinolytic, are permissive or predispositional to low level activation of coagulation in CFS/FM, there should be an increased prevalence of inheritable abnormalities in the patient population as compared with the general population.

(Null Hypothesis: There is no difference in inheritable abnormality prevalence rates between CFS/FM patients and the general population.)

Procoagulant Genetic Factors in CFS/FM Procoagulant Genetic Factors in CFS/FM and Related Chronic Illnessesand Related Chronic Illnesses

Hereditary and Acquired Hypercoagulability States Hereditary and Acquired Hypercoagulability States Hereditary

Antithrombin (fka antithrombin III)Protein CProtein SProthrombin (Factor II), G20210A polymorphismActivated Protein C Resistance (Factor V Leiden)Lipoprotein (a)Plasminogen Activator Inhibitor 1 (PAI-1)

Acquired Antiphospholipid Syndrome & SubgroupsCFS / FM

Procoagulant Genetic Factors in CFS/FM Procoagulant Genetic Factors in CFS/FM and Related Chronic Illnessesand Related Chronic Illnesses

7 7 ProcoagulantProcoagulant Genetic Factors TestedGenetic Factors Tested

Gen. Pop. Abnl RatesThrombophilic Antithrombin (AT) 0.02 to 0.5% Protein C (PC) 0.2 to 0.5% Protein S (PS) 0.2 to 0.5% Prothrombin (F2) 2 to 5% Act. Protein C Resistance (APCR) 5 to 7%Hypofibrinolytic Lipoprotein (a) [Lp(a)] 5% Plasminogen Act. Inh. 1 (PAI-1) 2%

Total: ~20%

Low Level Activation of Coagulation in Low Level Activation of Coagulation in Chronic Fatigue Syndrome and FibromyalgiaChronic Fatigue Syndrome and Fibromyalgia

-- Genetic Procoagulant FactorsGenetic Procoagulant Factors

CFS / HHV6 Cohort: Sequential Case AscertainmentCFS / HHV6 Cohort: Sequential Case Ascertainment

1. Sequential patients, one clinical site (N=45)

2. Patients assigned as CFS by CDC/NIH criteria.

3. Patients positive for HHV6 by blood culture.

4. All tested with ISAC and HTRP panels.

5. HTRP abnormality rates compared with general population rates.

Overall 84.4% of the patient cohort was positive for one or more of these markers.

Ref: Harrison, et al. Poster, AACFS 7th International Conference, 2004

Procoagulant Genetic Factors in CFS/FM Procoagulant Genetic Factors in CFS/FM and Related Chronic Illnessesand Related Chronic Illnesses

Present Study: Database MiningPresent Study: Database MiningDatabase of Activation of Coagulation (ISAC) and Hereditary Thrombosis Risk Panel (HTRP) scores in Chronic Illness patients (less well defined than smaller, specific CFS/FM cohorts).

582 adults (women/men = 2.3)

ISAC Score: One point per each abnormal result of FIB, F1+2, TAT, SFM, or Plt Act.

HTRP Score: One point per each abnormal result of AT, PC, PS, F2, APCR, LP(a), or PAI-1.

Genetic Procoagulant Factors in CFS/FM Genetic Procoagulant Factors in CFS/FM & Related Chronic Illnesses& Related Chronic Illnesses

Hereditary Thrombosis Risk Panel (HTRP) Score vs. Immune Hereditary Thrombosis Risk Panel (HTRP) Score vs. Immune System Activation of Coagulation (ISAC) Score (N=582)System Activation of Coagulation (ISAC) Score (N=582)

ISAC HTRP Score --> HTRP +ve HTRP +veScore 0 1 2 3 4 N 1 or more Rate

0 15 20 4 0 0 39 24 0.61541 50 57 12 3 0 122 72 0.59022 66 76 40 8 0 190 124 0.65263 34 77 45 11 1 168 134 0.79764 11 25 19 2 1 58 47 0.81035 2 1 2 0 0 5 3 0.6000N 178 256 122 24 2 582 404 0.6942

ISAC Neg 65 77 16 3 0 161ISAC Pos 113 179 106 21 2 421Pos Rate 0.635 0.699 0.869 0.875 1.000 0.723

Procoagulant Genetic Factors in CFS/FM and Related Chronic IllneProcoagulant Genetic Factors in CFS/FM and Related Chronic Illnessessses

Observed Abnormality RatesObserved Abnormality RatesCFS/FM Pts CFS / CI Rel Risk

Gen. Pop. HHV6 Cohort Database Ratio Abnl Rates (N=45) (N=582) (CI / Gen. Pop.)

Thrombophilic factorsAT 0.02 to 0.5% 0.0% 1.2% 2.42.4PC 0.2 to 0.5% 0.0% 1.2% 2.42.4PS 0.2 to 0.5% 6.7% 4.8% ~10~10F2 2 to 5% 42.2% 36.1% 7.27.2

APCR 5 to 7% 6.7% 6.7% 1Hypofibrinolytic factors

Lp (a) 5% 33.3% 32.5% 6.56.5PAI-1 2% 26.7% 17.5% 8.88.8

Genetic Procoagulant Factors in CFS/FM & Related Chronic IllnessGenetic Procoagulant Factors in CFS/FM & Related Chronic Illnesseses

Conclusions (N=582)Conclusions (N=582)1. A concerted / polygenic effect was observed in that overall

65.2% of the database cohort was positive for one or more of the procoagulant genetic markers [gpf=20%, OR=3.2], 52.9% for two or more [gpf=4%, OR=13.2], and 31.6% for 3 or more [gp<1%, OR=39.5].

2. Positivity for the factors cited represents a probable permissive or predispositional condition, yet the incidence of CFS/FM is far lower; thus, environmental factors.

3. Distinguishing thrombophilic and hypofibrinolytic procoagulant factors may help to guide therapy.

4. Patients with normal results for these seven markers may warrant testing for rarer inheritable disorders.

5. Testing family members may help assess their risks of developing CFS/FM as well as TED.

Soluble Fibrin Monomer (SFM)Soluble Fibrin Monomer (SFM) Coag Activation

Multiple Etiologies of ActivationMultiple Etiologies of ActivationViruses-HHV6, CMV, EBV

Bacteria -Mycoplasma, Chlamydia

Immune System Activation

(IIa) Coag Gene Defect

Inflammation

Fibrin Deposition &Fibrin Deposition & Tissue Ischemia / Hypoxia Tissue Ischemia / Hypoxia --Clinical symptoms in Chronic Fatigue SyndromeClinical symptoms in Chronic Fatigue Syndrome

Vaccine contaminants?Toxins, allergens?Parasites?

Yeast -Candida

Thrombin

Antiphospholipid Ab,Anti-B2GP1 Ab

+FibrinogenProthrombin F1+2

Thrombin-AntithrombinPlatelet Activation