Report
Cutaneous and Plasma Fibrinolytic Activity in Systemic Sclerosis Evidence of Normal Plasminogen Activation M. Matucci-Cerinic, M.D., T. Lotti, M.D., A. Lombardi, M.D., A. Pignone, M.D., F. lannone, M.D., E. Beneforti, S. Cinotti, B.D., M. Morfini, M.D., and M. Caignoni, M.D.
Abstract: Eleven patients with systemic sclerosis (SSc) were jury, arteriolar thrombosis, occlusion, and tissue ischstudied for plasma and cutaneous librinolytic activity, residual aemia).^ (potential fibrinolysis) fibrinolytic activity (FA) of the dermal Fibrinolysis is a physiological activity related to vessels that is related to the endothelial storage of plasmino- the activation of the plasma zymogen plasminogen to gen activators that become available due to particular stimuli the wide spectrum proteinase plasmin, which plays a such as intradermic injection of histamine, and the serum key role in dissolving thrombi occuring in the vascular levels of circulating von Willebrand antigen, antithrombin III, tree. The fibrinolytic system may be defined as "the plasminogen, ^-thromboglobulin, and platelet aggregate ratio enzymatic breakdown of fibrin to fragments that are (PAR). Cutaneous FA (autohistographic fibrin film method) apno longer able to form a coherent fibrin net."* peared normal or Increased in non-affected skin, normal In Fibrinolysis in normal human skin is subsequent lesional skin, and increased after intradermal (i.d.) injection of to the release of plasminogen activators (mainly tissue0.1 ml of 0.01% histamine. Monoclonal antibodies directed against the catalytic site of tissue type plasminogen activator type plasminogen activator) from the dermal venules.' completely blocked the fibrinolytic activity, while anti-urokiFibrinolysis has been investigated in SSc and the nase antibodies did not abolish the lysis areas. Plasmatic FA, plasma fibrinolytic activity (PFA) has been reported to euglobulin lysis time test, (ELT) and the levels of 13-tfirombog- be increased," normal,' or decreased.'""'" lobulin resulted similar to the controls. A significant increase in In our previous studies on cutaneous fibrinolytic von Willebrand Factor VIII antigen (but not of Factor VIII coaguactivity (CFA) in SSe and in motphea, a consistent FA lant) was observed in the patients (p < 0.01). Platelet aggreof the dermal vessels was evident and no impairment gate ratio, levels of plasma plasminogen and Antithrombin III in CFA was found in both disease.'^ These conflicting showed a significant difference (p < 0.01) when compared results induced us to study the following in a selected with the control subjects. Data suggest that primary injured microvessels in SSc are likely to be arteriolae while venulae group of 11 patients with SSc: 1) the extent of the could be affected by secondary hypoxia due to the arteriolar anatomical sites where plasminogen activators (PAs) damage with consequent release of tissue type plasminogen are released in the SSc skin; 2) the residual endothelial activator. Therefore, the authors suggest that the fibrinolytic FA (potential fibrinolysis) of the dermal vessels (that is potential is maintained in SSc and that the fibrinolytic therapy related to the endothelial storage of plasminogen actishould not be used in all patients with SSc but only in those vators which become available due to particular stimcases with documented exhaustion of plasmatic and/or cuta- uli such as intradermic injection of histamine); 3) the neous FA.
The pathogenesis of SSc is still unknown. Recently, it has been hypothesized that SSc is a microcirculatory disease'-^ and that a circulating cytotoxic factor, directed against endotheliocytes,-''' could account for the pathological vascular changes in SSc (endothelial in-
From the Departments of Clinical Medicine IV, Pathology, Dermatology 1, and Hematology, Universita' di Firenze, Firenze, Italy. Address correspondence to: M. Matucei-Cerinic, M.D., Via P. Thouar, 18, 50122 Firenze, Italy. 644
PFA, evaluated with ELT test; and 4) the serum levels of some haematological parameters that might test the extent of endothelial derangement (von Willebrand Factor antigen), the antithrombin activity (antithrombin III), the production of the zymogen (plasminogen) and the platelet activation (|8-thromboglobulin) and aggregation (platelet aggregate ratio). Materials and Methods Eleven patients (10 women, 1 man, range, 14-64 years of age) with SSc, all in the advanced phase of the disease, were selected and entered the study. All fulfilled the November 1990, Vol. 29, No. 9
No. 9
Normal Plasminogen Activation in Scleroderma • Matucci-Cerinic et al.
ARA cdteria for the classification of SSc." Patients discontinued all medications 2 weeks before entering the study. Venous blood samples were drawn always at the same time (9:00 a.m.) in the morning from the antecubital vein without venous stasis, of patients and of 11 control subjects (at rest and fasting) matched for sex and age. Sterile and siliconized needles (Butterfly 2tG, Abbott, Queenborough, Kent, UK) and appropriate Vacutainer tubes (Becton Dickinson, Meylan Cedex, France), placed in melting ice when required, were used to centrifuge at 4°C and 2000 G the blood, and collect the platelet poor plasma. Plasma aliquots were stored at -70°C up to time of assays, generally for no more than 15 days. The following assays were performed: 1) Factor VIII:C activity (FVIILC) was assayed by one stage clotting time method based on ellagic acid activated partial thromboplastin time (aPTT)'^; 2) Von Willebrand factor antigen (vWF:Ag) was assayed by Laurell electroimmunoassay'*; 3) Antithrombin III (AT III) was measured with amidolytic method with Coatest kit (KabiVitrum, Stockholm, Sweden)"; 4) Plasma )3-thromboglobulin ()3-TG) was assayed on platelet-poor plasma, collected with the anticoagulant mixture supplied by the manufacturer (Radiochemical Centre, Amersham, England), in accordance with Bolton et al.^"; 5) Plasminogen was assayed with an amidolytic method^'; 6) Platelet aggregate ratio ie, the ratio between platelet count collected in EDTA and formalin 4% and in EDTA alone according to Wu and Hoak^^; and 7) The PFA was assessed in patients and in controls by the ELT test, performed according to the method of Nilsson et al.'^' In these 11 patients with SSc, after informed consent, we obtained samples by punch biopsies, from 1) affected skin (dorsum of the proximal phalanx of the II finger of the left hand); 2) apparently uninvolved skin (volar aspect of the right arm); and 3) uninvolved histamine-injected skin (volar aspect of the right arm). The biopsies were taken 2 hours after the injection of O.t ml of 0.01% histamine hydrochloride. We took advantage of the intradermic histamine injection (capable of releasing endothelial PAs in tissue'^''"^'' into the uninvolved skin, to evaluate the potential cutaneous FA, that is, the storage of PAs, which can become available due to particular stimuli.^''"^^ To evaluate CFA, the biopsies were studied by Todd's histochemical fibrin slides technique,^'-^^ as modified by our group, "•^''••* so that thickness of the fibrin film and of the tissue slices were constant. Fibrinolytically active sites appeared under microscopic observations as clear punched out areas of lysis around the dermal vessels in the opaque fibrin film. The specimens were blindly analyzed at the microscope by the same physician (TL) that estimated the degree of FA by comparing the lysis areas with those of normal skin. The results were tabulated according to a scale graphically expressed as 0, (+), (++), and (+++), which represent both the size and number of the fibrin lysis areas at the perivascular level in the dermis. 0 indicates the cases
645
without CFA; (4-) indicates the cases with 50% less FA than that found in healthy controls: (++) indicates cases with FA equal to that of the control group; and (+++) those with FA more than 50% higher than control group. This, of course, was an arbitrary method of evaluation, but it can be considered valid since both the tissue and fibrin film thicknesses were uniform in all tests performed. Sera and skin biopsies, taken from the volar aspect of the right arm of 11 healthy donors, matched for sex and age were used as controls. In order to distinguish between urokinase (UK) and tissue-type (tPA) PAs dependent FA, we used the method of apposition on the skin sections of specific monoclonal antibodies (monoclonal antibodies, Monozyme, Lyngby, Denmark) to UK and to tPA^^ in 11 specimens of lesionai skin from subjects with SSc and in 11 controls. A solution of 2 ml of the specific monoclonal antibody (50 /ig/ml) is put on the tissue sections at room temperature. Five minutes later, the fibrin film is placed on the section, which is processed as above reported. Both antibodies (Mouse IgGt subclass) are purified by affinity chromatography with protein A. The specific antibodies react only with their substrate (respectively, UK and tPA) and inhibit their specific substrate enzyme activity against plasminogen, thus allowing a determination of the UK or tPA part of the overall cutaneous plasminogen activator activity. With this device, specific tPA and/or UK dependent FA can be selectively inhibited in the skin in the anatomic sites where it is produced so that cutaneous tPA and UK activity can be assessed individually.'•^'•^* Comparison measurements of the haematological parameters of SSc patients and controls were made using the Student's t-test and, because of the high SD, the U-Mann Whitney test. The relationship between laboratory data and clinical characteristics was examined by linear regression analysis.
Resuits
The results of CFA and PFA concerning SSc patients are outlined respectively in Tables 1 and 2. Cutaneous Fibrinolytic Activity
In lesionai skin, all the samples showed a normal CFA (++), which appeared as areas of lysis centered around microvessels. Monoclonal antibodies anti-UK did not abolish the lysis areas. In all the samples, we found that CFA was completely blocked by anti-tPA antibodies. In apparently healthy skin, CFA was increased (+4-+) in 5/11 and normal (-I-+) in 6/11 non-histamine injected cases. In histamine injected skin, CFA was increased (+++) in all cases tested. In all the cases, the test with plasminogen free-film showed a lack of FA. Using monoclonal antibodies directed against the cata-
Vol. 29
International Journal of Dermatology • November 1990
646
Table 1. Cutaneous Fibrinolytie Activity (CFA) Evaluated with Autohistographic Technique Modified Using Monoclonal Antibodies Against Tissue-Type Plasminogen Activator (tPA) and Urokinase (UK) in 11 Patients with Systemic Sclerosis Plasm in ogen -free Film Test CFA
Taken from apparently uninvolved skin
CFA Present
CFA Absent
Specimens Without histamine injection With histamine injection
—
—
Plasminogen Activator Activity after Apposition of Monoclonal Antibodies
tl
6
Anti-UK
Anti-tPA
FA is not affected
FA is completely abolished FA is completely abolished
FA is not affected FA is not affected
11
Taken from sclerodermic skin
lytic site of tPA and of UK, we observed that the areas of lysis were aholished only after apposition of the anti-tPA antibodies while anti-UK antibodies had no visible effect on the areas of lysis. Plasma Fibrinolytie Activity
The mean blood levels of vWF:Ag, FVI1I:C, AT III, /3-TG, Plasminogen, ELT, and PAR in both SSc and controls are shown in Table 2. Euglobulin lysis time did not show any statistical difference from the control group. A significant increase of vWF:Ag (but not of FVIII.C) was observed in all SSc patients. The vWF:Ag/FVIII:C ratio did not show any difference between patients and normal control. Likewise, Pthromboglohulin did not show any statistical differences with the control group. Values of PAR, AT III,
FA is completely abolished
and serum plasminogen levels showed significant differences when compared with the control subjects. There was no correlation between the laboratory variables (vWF:Ag, FVIIIiC, PAR, ELT, AT III, /3thrornboglobulin and plasminogen) and patient age, disease duration, digital ulcers, or number of organ systems involved (data not shown). No appreciable statistical correlation among laboratory variables was found. Discussion
It has been suggested that the vessel involvement in SSc may give rise to coagulation abnormalities with widespread fibrin deposition. For this reason, the fibrinolytie pathway has been studied in SSc. Cunliffe and Menon" showed a decrease of PFA in 12 patients af-
Table 2. The Mean {± SD) Blood Levels of von Willebrand Factor Antigen (vWF:Ag), Factor VIII Coagulant (FVIIhC), Ratio vWF:Ag/FVIII:C (vWFiAg/C), Platelet Aggregate Ratio (PAR), Euglobulin Lysis Time Test (ELT), Antithrombin III (AT III), Beta Thromboglobulin (0-TG), and Plasminogen in 11 Patients with Systemic Scierosis (SSc) and 11 Control Subjects SSc vWF:Ag (%) FV111:C (%) Ratio vWF:Ag/C PAR ELT (hr) AT 111% fi-TG (ng/ml) Plasminogen (%) NS: not significant.
185.5 ±29.7 164.6 ± 24.1 0.96 ± 0 . 1 6 0.73 ±0.054 5.54 ± 0 . 6 3 119 ±6.57 57.18 ± 11.45 106.27 ± 6.65
Controls
Student's t-Test P
U-Mann Whitney Test P
108.9 ± 8.1 115.7 ± 6 . 5 0.95 ± 0.07 0.91 ±0.047 4.45 ± 0.43 101 ± 2 . 2 5 43.27 ± 4 . 5 3 88.27 ± 3.24
Cutaneous and Plasma Fibrinolytic Activity in Systemic Sclerosis Evidence of Normal Plasminogen Activation M. Matucci-Cerinic, M.D., T. Lotti, M.D., A. Lombardi, M.D., A. Pignone, M.D., F. lannone, M.D., E. Beneforti, S. Cinotti, B.D., M. Morfini, M.D., and M. Caignoni, M.D.
Abstract: Eleven patients with systemic sclerosis (SSc) were jury, arteriolar thrombosis, occlusion, and tissue ischstudied for plasma and cutaneous librinolytic activity, residual aemia).^ (potential fibrinolysis) fibrinolytic activity (FA) of the dermal Fibrinolysis is a physiological activity related to vessels that is related to the endothelial storage of plasmino- the activation of the plasma zymogen plasminogen to gen activators that become available due to particular stimuli the wide spectrum proteinase plasmin, which plays a such as intradermic injection of histamine, and the serum key role in dissolving thrombi occuring in the vascular levels of circulating von Willebrand antigen, antithrombin III, tree. The fibrinolytic system may be defined as "the plasminogen, ^-thromboglobulin, and platelet aggregate ratio enzymatic breakdown of fibrin to fragments that are (PAR). Cutaneous FA (autohistographic fibrin film method) apno longer able to form a coherent fibrin net."* peared normal or Increased in non-affected skin, normal In Fibrinolysis in normal human skin is subsequent lesional skin, and increased after intradermal (i.d.) injection of to the release of plasminogen activators (mainly tissue0.1 ml of 0.01% histamine. Monoclonal antibodies directed against the catalytic site of tissue type plasminogen activator type plasminogen activator) from the dermal venules.' completely blocked the fibrinolytic activity, while anti-urokiFibrinolysis has been investigated in SSc and the nase antibodies did not abolish the lysis areas. Plasmatic FA, plasma fibrinolytic activity (PFA) has been reported to euglobulin lysis time test, (ELT) and the levels of 13-tfirombog- be increased," normal,' or decreased.'""'" lobulin resulted similar to the controls. A significant increase in In our previous studies on cutaneous fibrinolytic von Willebrand Factor VIII antigen (but not of Factor VIII coaguactivity (CFA) in SSe and in motphea, a consistent FA lant) was observed in the patients (p < 0.01). Platelet aggreof the dermal vessels was evident and no impairment gate ratio, levels of plasma plasminogen and Antithrombin III in CFA was found in both disease.'^ These conflicting showed a significant difference (p < 0.01) when compared results induced us to study the following in a selected with the control subjects. Data suggest that primary injured microvessels in SSc are likely to be arteriolae while venulae group of 11 patients with SSc: 1) the extent of the could be affected by secondary hypoxia due to the arteriolar anatomical sites where plasminogen activators (PAs) damage with consequent release of tissue type plasminogen are released in the SSc skin; 2) the residual endothelial activator. Therefore, the authors suggest that the fibrinolytic FA (potential fibrinolysis) of the dermal vessels (that is potential is maintained in SSc and that the fibrinolytic therapy related to the endothelial storage of plasminogen actishould not be used in all patients with SSc but only in those vators which become available due to particular stimcases with documented exhaustion of plasmatic and/or cuta- uli such as intradermic injection of histamine); 3) the neous FA.
The pathogenesis of SSc is still unknown. Recently, it has been hypothesized that SSc is a microcirculatory disease'-^ and that a circulating cytotoxic factor, directed against endotheliocytes,-''' could account for the pathological vascular changes in SSc (endothelial in-
From the Departments of Clinical Medicine IV, Pathology, Dermatology 1, and Hematology, Universita' di Firenze, Firenze, Italy. Address correspondence to: M. Matucei-Cerinic, M.D., Via P. Thouar, 18, 50122 Firenze, Italy. 644
PFA, evaluated with ELT test; and 4) the serum levels of some haematological parameters that might test the extent of endothelial derangement (von Willebrand Factor antigen), the antithrombin activity (antithrombin III), the production of the zymogen (plasminogen) and the platelet activation (|8-thromboglobulin) and aggregation (platelet aggregate ratio). Materials and Methods Eleven patients (10 women, 1 man, range, 14-64 years of age) with SSc, all in the advanced phase of the disease, were selected and entered the study. All fulfilled the November 1990, Vol. 29, No. 9
No. 9
Normal Plasminogen Activation in Scleroderma • Matucci-Cerinic et al.
ARA cdteria for the classification of SSc." Patients discontinued all medications 2 weeks before entering the study. Venous blood samples were drawn always at the same time (9:00 a.m.) in the morning from the antecubital vein without venous stasis, of patients and of 11 control subjects (at rest and fasting) matched for sex and age. Sterile and siliconized needles (Butterfly 2tG, Abbott, Queenborough, Kent, UK) and appropriate Vacutainer tubes (Becton Dickinson, Meylan Cedex, France), placed in melting ice when required, were used to centrifuge at 4°C and 2000 G the blood, and collect the platelet poor plasma. Plasma aliquots were stored at -70°C up to time of assays, generally for no more than 15 days. The following assays were performed: 1) Factor VIII:C activity (FVIILC) was assayed by one stage clotting time method based on ellagic acid activated partial thromboplastin time (aPTT)'^; 2) Von Willebrand factor antigen (vWF:Ag) was assayed by Laurell electroimmunoassay'*; 3) Antithrombin III (AT III) was measured with amidolytic method with Coatest kit (KabiVitrum, Stockholm, Sweden)"; 4) Plasma )3-thromboglobulin ()3-TG) was assayed on platelet-poor plasma, collected with the anticoagulant mixture supplied by the manufacturer (Radiochemical Centre, Amersham, England), in accordance with Bolton et al.^"; 5) Plasminogen was assayed with an amidolytic method^'; 6) Platelet aggregate ratio ie, the ratio between platelet count collected in EDTA and formalin 4% and in EDTA alone according to Wu and Hoak^^; and 7) The PFA was assessed in patients and in controls by the ELT test, performed according to the method of Nilsson et al.'^' In these 11 patients with SSc, after informed consent, we obtained samples by punch biopsies, from 1) affected skin (dorsum of the proximal phalanx of the II finger of the left hand); 2) apparently uninvolved skin (volar aspect of the right arm); and 3) uninvolved histamine-injected skin (volar aspect of the right arm). The biopsies were taken 2 hours after the injection of O.t ml of 0.01% histamine hydrochloride. We took advantage of the intradermic histamine injection (capable of releasing endothelial PAs in tissue'^''"^'' into the uninvolved skin, to evaluate the potential cutaneous FA, that is, the storage of PAs, which can become available due to particular stimuli.^''"^^ To evaluate CFA, the biopsies were studied by Todd's histochemical fibrin slides technique,^'-^^ as modified by our group, "•^''••* so that thickness of the fibrin film and of the tissue slices were constant. Fibrinolytically active sites appeared under microscopic observations as clear punched out areas of lysis around the dermal vessels in the opaque fibrin film. The specimens were blindly analyzed at the microscope by the same physician (TL) that estimated the degree of FA by comparing the lysis areas with those of normal skin. The results were tabulated according to a scale graphically expressed as 0, (+), (++), and (+++), which represent both the size and number of the fibrin lysis areas at the perivascular level in the dermis. 0 indicates the cases
645
without CFA; (4-) indicates the cases with 50% less FA than that found in healthy controls: (++) indicates cases with FA equal to that of the control group; and (+++) those with FA more than 50% higher than control group. This, of course, was an arbitrary method of evaluation, but it can be considered valid since both the tissue and fibrin film thicknesses were uniform in all tests performed. Sera and skin biopsies, taken from the volar aspect of the right arm of 11 healthy donors, matched for sex and age were used as controls. In order to distinguish between urokinase (UK) and tissue-type (tPA) PAs dependent FA, we used the method of apposition on the skin sections of specific monoclonal antibodies (monoclonal antibodies, Monozyme, Lyngby, Denmark) to UK and to tPA^^ in 11 specimens of lesionai skin from subjects with SSc and in 11 controls. A solution of 2 ml of the specific monoclonal antibody (50 /ig/ml) is put on the tissue sections at room temperature. Five minutes later, the fibrin film is placed on the section, which is processed as above reported. Both antibodies (Mouse IgGt subclass) are purified by affinity chromatography with protein A. The specific antibodies react only with their substrate (respectively, UK and tPA) and inhibit their specific substrate enzyme activity against plasminogen, thus allowing a determination of the UK or tPA part of the overall cutaneous plasminogen activator activity. With this device, specific tPA and/or UK dependent FA can be selectively inhibited in the skin in the anatomic sites where it is produced so that cutaneous tPA and UK activity can be assessed individually.'•^'•^* Comparison measurements of the haematological parameters of SSc patients and controls were made using the Student's t-test and, because of the high SD, the U-Mann Whitney test. The relationship between laboratory data and clinical characteristics was examined by linear regression analysis.
Resuits
The results of CFA and PFA concerning SSc patients are outlined respectively in Tables 1 and 2. Cutaneous Fibrinolytic Activity
In lesionai skin, all the samples showed a normal CFA (++), which appeared as areas of lysis centered around microvessels. Monoclonal antibodies anti-UK did not abolish the lysis areas. In all the samples, we found that CFA was completely blocked by anti-tPA antibodies. In apparently healthy skin, CFA was increased (+4-+) in 5/11 and normal (-I-+) in 6/11 non-histamine injected cases. In histamine injected skin, CFA was increased (+++) in all cases tested. In all the cases, the test with plasminogen free-film showed a lack of FA. Using monoclonal antibodies directed against the cata-
Vol. 29
International Journal of Dermatology • November 1990
646
Table 1. Cutaneous Fibrinolytie Activity (CFA) Evaluated with Autohistographic Technique Modified Using Monoclonal Antibodies Against Tissue-Type Plasminogen Activator (tPA) and Urokinase (UK) in 11 Patients with Systemic Sclerosis Plasm in ogen -free Film Test CFA
Taken from apparently uninvolved skin
CFA Present
CFA Absent
Specimens Without histamine injection With histamine injection
—
—
Plasminogen Activator Activity after Apposition of Monoclonal Antibodies
tl
6
Anti-UK
Anti-tPA
FA is not affected
FA is completely abolished FA is completely abolished
FA is not affected FA is not affected
11
Taken from sclerodermic skin
lytic site of tPA and of UK, we observed that the areas of lysis were aholished only after apposition of the anti-tPA antibodies while anti-UK antibodies had no visible effect on the areas of lysis. Plasma Fibrinolytie Activity
The mean blood levels of vWF:Ag, FVI1I:C, AT III, /3-TG, Plasminogen, ELT, and PAR in both SSc and controls are shown in Table 2. Euglobulin lysis time did not show any statistical difference from the control group. A significant increase of vWF:Ag (but not of FVIII.C) was observed in all SSc patients. The vWF:Ag/FVIII:C ratio did not show any difference between patients and normal control. Likewise, Pthromboglohulin did not show any statistical differences with the control group. Values of PAR, AT III,
FA is completely abolished
and serum plasminogen levels showed significant differences when compared with the control subjects. There was no correlation between the laboratory variables (vWF:Ag, FVIIIiC, PAR, ELT, AT III, /3thrornboglobulin and plasminogen) and patient age, disease duration, digital ulcers, or number of organ systems involved (data not shown). No appreciable statistical correlation among laboratory variables was found. Discussion
It has been suggested that the vessel involvement in SSc may give rise to coagulation abnormalities with widespread fibrin deposition. For this reason, the fibrinolytie pathway has been studied in SSc. Cunliffe and Menon" showed a decrease of PFA in 12 patients af-
Table 2. The Mean {± SD) Blood Levels of von Willebrand Factor Antigen (vWF:Ag), Factor VIII Coagulant (FVIIhC), Ratio vWF:Ag/FVIII:C (vWFiAg/C), Platelet Aggregate Ratio (PAR), Euglobulin Lysis Time Test (ELT), Antithrombin III (AT III), Beta Thromboglobulin (0-TG), and Plasminogen in 11 Patients with Systemic Scierosis (SSc) and 11 Control Subjects SSc vWF:Ag (%) FV111:C (%) Ratio vWF:Ag/C PAR ELT (hr) AT 111% fi-TG (ng/ml) Plasminogen (%) NS: not significant.
185.5 ±29.7 164.6 ± 24.1 0.96 ± 0 . 1 6 0.73 ±0.054 5.54 ± 0 . 6 3 119 ±6.57 57.18 ± 11.45 106.27 ± 6.65
Controls
Student's t-Test P
U-Mann Whitney Test P
108.9 ± 8.1 115.7 ± 6 . 5 0.95 ± 0.07 0.91 ±0.047 4.45 ± 0.43 101 ± 2 . 2 5 43.27 ± 4 . 5 3 88.27 ± 3.24
