Protein S Deficiency in Men With Long-Term Human

Protein S Deficiency in Men With Long-Term Human Immunodeficiency Virus Infection

By Claire Philipp Stahl, Carol S. Wideman, Thomas J. Spira, Elizabeth C. Haff, Gwendolyn J. Hixon, and Bruce L. Evatt

Decreases in protein S levels have recently been reported in some human immunodeficiency virus (HIV)-infected patients. To examine predisposing factors, 25 men randomly selected from a long-term study of HIV-infected patients were studied. The minimum mean duration of HIV sero- positivity in this group was 106.6 months (range 1 5 to 143 months). No patients were anticoagulated at the time of the study. Three of the 25 randomly selected patients gave a history of thrombosis, in each instance occurring afterthe onset of HIV positivity. Two of the 3 patients with thrombosis had more than one episode. Coagulation studies showed that 3 of 3 (1 00%) of the patients with thrombosis and 16 of 22 (72.7%) of those without previous thrombosis had decreased free protein S. Mean-free and total protein S levels were statistically lower for HIV-infected patients with and without previous thrombosis compared with healthy male controls. C4b-binding protein was not in-

ROTEIN S IS A vitamin K-dependent plasma protein P that serves as a cofactor for activated protein C.’ Con- genital deficiencies in protein S predispose to thromboembolic complication^.^-^ Hereditary protein S deficiency has been estimated to have a prevalence in the general population of 1 in 29,0005 and to be found in 5% to 8% of individuals with unexplained thrombosis detected at a young age in the ab- sence of known predisposing factors. Normally, approximately 30% to 40% of protein S is free in the plasma and approximately 60% to 70% is bound to C4b-binding protein (C4bBP).6 The latter is functionally inactive as a cofactor for activated protein C . Hereditary reductions in free protein S, caused by either an abnormal distribution between free protein S and inactive protein S bound to C4bBP or caused by reductions in total protein S, increase the risk for thrombosis.’

Acquired decreases in protein S levels occur in pregnancy: with oral contraceptive use: and during warfarin therapy,” and are associated with disseminated intravascular coagulation,” liver disease,” nephrotic syndrome,12,13 and systemic lupus erythemato~us.’~ Decreases in protein S levels also have been reported in some human immunodeficiency virus (HIV)-infected patients.” However, whether acquired reductions in protein S levels predispose individuals to thrombosis is currently not known. In congenital protein S deficiency the probability of developing thrombosis by age 27 is 50% and increases to 70% by age 35.16 Because of these findings, we evaluated retrospectively the clinical implications of the protein S status of a group of men with long-term HIV infection. The results suggest that these individuals may be predisposed to thrombotic complications.

MATERIALS AND METHODS

Twenty-seven men with HIV infection (mean age 38.6 f 5.6 years) and 28 healthy male controls (mean age 40 * 7.9 years) were studied. All subjects gave informed consent. Venous blood was collected in 1/10 vol ofO. 109 mol/L sodium citrate. Platelet- poor plasma was obtained by centrifugation and frozen at -70°C until tested. Twenty-five men with HIV disease were randomly selected from a previously established long-term prospective study of HIV-associated lymphadenopathy. An additional two men with HIV

Patient population.

creased in study patients with decreased protein S levels. Decreases in protein S levels did not correlate with CD4+ cell levels, CDC class, p24 antigen positivity, zidovudine (AZT) use, or Pneumocystis carinii prophylaxis. The duration of disease statistically correlated with decreases in protein S levels (r = .37, P < .05). A linear correlation existed between increasing lgG anticardiolipin antibody levels and decreasing free protein S antigen (r = .67, P < .005). This study shows that protein S deficiency is common in long- term HIV-infected patients and is caused by a decrease in the free protein, rather than by changes in the bound complex. The data suggest that protein S deficiency is not correlated with HIV disease severity but may predispose patients to thromboembolic complications. This is a US government work. There are no restrictions on its use.

disease were referred for evaluation of previous thrombosis. Their results are presented separately and are not included in the analysis of data. None of the individuals were on anticoagulants or had active thrombosis at the time of study. None of the patients had laboratory or clinical evidence of liver or kidney disease. All patients were am- bulatory at the time of study and there was no evidence of acute intercurrent infection. A minimum mean duration of HIV seropos- itivity for the 25 randomly selected HIV-infected men was 106.6 months (range 15 to 143). Their mean CD4+ cell level was 226/mm3 (range 29 to 653) (Table 1). Of the 25 men, 13 males with HIV disease were taking zidovudine (AZT) at the time of the study, 4, dideoxycytidine (DDC), and 1, dideoxyinosine (DDI). Twelve patients were on medications for Pneumocystis prophylaxis, including pent- amidine (8), trimethoprim-sulfamethoxazole (4), and dapsone ( I ) . The control population was not on medications.

Protein C antigen and total protein S antigen were measured with the Laurel1 electroimmunoassay as previously

Free protein S antigen was determined by polyethylene- glycol pre~ipitation,~ followed by electroimmunoassay to measure the free protein S. The protein S concentration in plasma was assessed using purified human protein S (American Diagnostica Inc, Green- wich, CT) as a reference standard, and the molarity was calculated using a protein S molecular weight of 75,000.’8 Levels of protein S in the standard were measured by determining its amino acid concentration following 24-hour 6N HC1 hydrolysis in vacuo. The phenylthiocarbamoyl amino acids were analyzed by high performance liquid chromatography (HPLC) by the Waters Pico-Tag system (Mil-

Coagulation studies.

From the Division of HIV/AIDS, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department ofHealth and Human Services, Atlanta, GA.

Submitted April 16, 1992; accepted November 23, 1992. American Bioproducts Company (Parsippany, NJ/ provided the

C4bBP kits under Grant No. 91ER005. Address reprint requests to Claire Philipp Stahl, MD, Centers for

Disease Control, 1600 Clifton Road, 1-1378 (002). Atlanta, GA 30333. The publication costs of this article were defrayed in part by page

charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

This is a USgovernment work. There are no restrictions on its use. 0006-49 71/93/81 0 7-0028$0.00/0

Blood, Vol 81, No 7 (April 1). 1993: pp 1801-1807 1801

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1802 STAHL ET AL

Table 1. Immunologic, Clinical, and Coagulation Parameters for Study Patients With HIV

CD4, CD4:CD8 Months Free S Funct. S Total S vWF:AG 656- Ratio 0.7%- Since Thrombotic 5.5-10.2 63%- 18.1-32.0 44%- Fibrinogen

Patient 1801/pL 3.4% CDC Class Onset Events m/mL 144% rg/mL 140% 2.0-4.0 g/L

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mean Range

653 29

292 189 41

202 430 377

29 83

600 160 250 228 387 225 383 196 160 29 46

172 242 218

29 226

29-653

0.37 0.05 0.29 0.23 0.07 0.61 0.59 0.38 0.07 0.09 0.32 0.13 0.20 0.32 0.21 0.22 0.23 0.16 0.52 0.13 0.04 0.1 1 0.16 0.40 0.05 0.24

0.04-0.6 1

2 4C 1 4C2 3 3 3 3 3 4D 4C2 2 4C 1 3 3 3 4D 3 3 3 3 3 3 3 3 3

59 121 106 120 107 102 75

122 96

109 19

114 123 102 112 108 107 103 97

107 139 143 123 117 134 106.6

19-143

NONE MI, DVT NONE NONE NONE NONE NONE DVT, PE NONE NONE NONE NONE NONE NONE NONE NONE NONE NONE NONE DVT NONE NONE NONE NONE NONE

5.5 1.3 5.5 5.5 7.7 3.4 5.3 3.4 3.4 3.4 6.6 1.7 4.4 4.0 2.8 6.9 3.3 4.8 2.1 1.8 2.4 3.3 3.6 4.9 2.1 4.0

1.3-7.7

102 44

114 93 92 71 78 51 26 68

128 34 93 65 44 88 61 89 30 33 66 83

118 124 43 74

26-1 28

19.5 20.0 17.6 19.7 32.5 20.8 21.1 16.0 17.9 19.5 30.7 19.2 21.3 16.8 17.9 26.1 17.6 26.1 14.9 17.1 24.8 26.7 29.3 27.7 19.2 21.6

14.9-32.5

175 2.50 20 1 5.30

72 1.90 105 2.20 555 3.30

64 2.30 20 1 3.00 161 2.20 218 2.25 131 3.15 182 2.95 165 4.35 192 3.00 218 2.82

86 2.52 121 4.10 152 3.11 90 2.02 84 2.35

486 2.95 340 2.70 122 2.67 107 2.67 97 2.14

256 2.56 183 2.84

64-555 1.90-5.30

Patients were randomly selected from a previously established cohort of HIV-infected men (see Materials and Methods). Months since onset of

Abbreviations: MI, myocardial infarct; DVT, deep vein thrombosis; PE, pulmonary embolism. HIV positivity and/or onset of first symptoms are reported.

lipore/Water, Milford, MA). The concentration of total protein S in normal-pooled plasma from six men and six women corrected for anticoagulant dilution was determined to be 22.28 f 0.84 pg/mL, and of free protein S, 6.04 f 0.59 &mL. These values are consistent with previously reported concentrations'O.'s-'o when adjusted to the levels for undiluted plasma. Crossed immunoelectrophoresis of protein S was performed using the method of Laurell and McKay as previously described:' with a 1:428 dilution of protein S antiserum (American Diagnostica). Functional protein S was measured with Staclot Protein S kits (Diagnostica Stago, Parsippany, NJ) using the ST4 coagulation instrument (Diagnostica Stago). The test measures the increase in activated partial thromboplastin time (APTT) performed on a mixture containing protein Sdeficient plasma, cephalin, test plasma, activated protein C, factor Va, kaolin, and calcium chlo- ride.*' Functional protein C was measured with the Stachrom Protein C kit (Diagnostica Stago). C4bBP antigen was measured with the Laurell electroimmunoassay using C4bBP agarose gel plates (Diag- nostica Stago) and with Liatest C4b-BP (Diagnostica Stago). The von Willebrand factor antigen (vWFAG) was measured by the Laurell electroimmunoassay using rabbit human-factor VIII-related antigen antiserum (Accurate Chemical and Scientific Corp, Westbury, NY). Fibrinogen was measured by the Clauss procedure (Dade, Miami, FL). Antithrombin I11 was determined on the ACA (DuPont, Wil- mington, DE) using bovine thrombin. Plasminogen was also assayed on the ACA. The tissue thromboplastin inhibition test (TTI)23 was modified by testing a 1: 1 mix of patient and normal plasma to correct for possible coagulation factor deficiencies. Factor I1 and X antigens were measured by the Laurell electroimmunoassay using prothrombin

rabbit antiserum (Behring Corp, La Jolla, CA, and Accurate Chemical and Scientific Cow) and factor X rabbit antiserum (Diagnostica Stago).

Discontinuous sodium dodecyl sulfate (SDS) electrophoresis was performed on 7.5%, IO%, or 10% to 15% gradient nondenaturing polyacrylamide gels at 15°C in a Phast System (Pharmacia LKB Biotechnology, Piscataway, NJ). Proteins were im- mediately electrotransferred to nitrocellulose (Hoefer Scientific In- struments, San Francisco, CA) or polyvinylidene difluoride (Millipore, Bedford, MA) in 25 mmol/L Tris, 192 mmol/L glycine, 20% (vol/ vol) methanol, pH 8.3, at 15°C in a PhastTransfer Semi-dry System (Pharmacia LKB Biotechnology) at 25 mA as limiting factor for 3 volt hours.

The membrane was blocked in bovine lacto transfer technique optimizer probed with protein S antisera (American Diagnostica, New York, NY), then incubated with anti-goat IgG horseradish peroxidase conjugate (Chemicon International, Temecula, CA). Molecular weight determinations were performed with pre- stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) stan- dards (Bio-Rad, Rockville Centre, NY).

IgG and IgM anticardiolipin antibodies were measured using a semi-quantitative in- direct enzyme-linked immunosorbent assay (ELISA) test kit (Reaads Medical Products, Inc, Westminster, CO).25 A standard curve was generated using standard sera provided by the Antiphospholipid Standardization Laboratory (University of Louisville, Louisville, KY). The anticardiolipin antibody concentration is expressed in standard units where 1 IgG phospholipid (GPL) and IgM phospholipid (MPL)

Immunoblot procedure.

Anticardiolipin antibodies and P24 antigen.




HIV AND PROTEIN S DEFICIENCY

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HIV without Thrombosis

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Control Males

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HIV with Thrombosis

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Fig 1. Free protein S antigen levels (A) and total protein S antigen levels (B) in male controls, HIV-infected men with previous thrombosis, and HIV-infected men without thrombosis are shown. Mean values for each group are indicated by a line. Free protein S values for both groups of HIV-infected men were significantly different from those for control men (P < .OOOl). Total protein S levels were significantly lower in both groups of HIV-infected males compared with male controls (HIV with thrombosis, P < .001; HIV without thrombosis, P < .02). Two additional HIV patients referred for thrombosis are shown with (A) but are not included in mean values or statistical analysis.

unit is equivalent to 1 pg/mL of an affinity-purified standard IgG or IgM. Positive values are reported as slightly, moderately, or highly positive. P24 antigens were performed using the Coulter P24 antigen kit (Coulter, Hialeah, FL).

Comparisons between groups were performed using the Wilcoxon rank sum test. Spearman’s rank correlation coef- ficient was used to assess correlations between two parameters. Results were considered statistically significant for P < .05.26

Statistical analysis.

RESULTS

Of the 25 HIV-infected men, 3 had a history of one or more previous episodes of thromboembolism (Table 1). Two out of 3 of the men with thrombosis had more than one episode. In addition to one myocardial infarct, there were four episodes of venous thrombosis, including one episode of pulmonary embolism documented by angiography and three episodes of deep vein thrombosis (DVT) documented by venogram ( 2 ) and Doppler studies (1). All episodes oc- curred after infection with HIV. Two patients had thrombosis within the first year after the diagnosis of HIV-associated lymphadenopathy. Of the 2 additional HIV-positive patients referred with a previous diagnosis of thrombosis, 1 patient

had two episodes of pulmonary emboli and an episode of DVT, and I patient had a pulmonary embolus and a DVT. All of these episodes were documented by lung scans or venograms.

In this study, various coagulation parameters associated with hypercoagulable states were examined. None of the 25 HIV-infected men had decreased protein C, antithrombin 111, plasminogen levels, or evidence of a lupus inhibitor as measured by tissue thromboplastin inhibition test. Fi- brinogen was increased in two HIV-infected men. Anti- cardiolipin antibodies, which have been associated with an increased frequency of thrombosis in patients with systemic lupus erythematosus*’ but not with H I V - d i s e a ~ e , ~ ~ - ~ ~ were also measured in this study. IgM anticardiolipin antibodies were strongly positive in 1 of 18 HIV-infected men, whereas IgG anticardiolipin antibodies were strongly positive in 3 of 24 and moderately positive in 14 of 24. vWF:AG was increased in 3 of 3 study patients with previous thrombosis and 12 of 29 patients with no previous history. Factor I1 and X antigen levels were normal in all study patients.




1804

130

120

STAHL ET AL

-

12 L

10

l1 1 11 - 10 -

- 9 :

a*: E

.E 6 - w .

Q .

I e 5 -

0 ' 1 1 " " " " ' 1

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Functional Protein S ("A normal) 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Functional Protein S ("A normal)

Fig 2. Correlation between functional protein S determinations and free protein S antigen levels in 25 study patients. A linear regression line is drawn. A significant correlation is seen between functional and free protein S determinations (r = .78, P < .005).

Protein S deficiency was common in the randomly selected group of HIV-infected men. Free protein S antigen was decreased in 3 of 3 (100%) randomly selected men with previous thrombosis and in 16 of 22 (72.7%) of those men without a history of thromboembolism. The HIV-infected men with a prior episode of thrombosis had a mean (? 1 SD) free protein S level of 2.2 f 1.1 pg/mL (range 1.3 to 3.4 pg/mL, n = 3) compared with a mean free protein S level of 4.2 ? 1.6 pg/ mL (range 1.7 to 7.7 pg/mL, n = 22) in HIV-infected men without a previous history of thrombosis. Male controls had a mean free protein S level of 7.9 f 1.2 pg/mL (range 5.0 to 10.7 pg/mL, n = 27). Mean values for both groups of HIV- infected men were significantly different from those for un- infected men (P < .0001) (Fig 1). Total protein S, which includes both bound and unbound protein S, was also significantly decreased in both groups of HIV-infected men, with mean levels of 17.7 f 2.1 pg/mL (range 16.0 to 20.0 pg/mL) in the three men with previous thrombosis ( P < .OO l), and 22.1 f 4.9 pg/mL (range 14.9 to 32.5 pg/mL, n = 22) in HIV-infected men without previous thrombosis (P < .02) compared with a mean level of 25.0 +. 3.2 pg/mL (range 18.1 to 32.0 pg/mL) in male controls (Fig I ) . Functional protein S determinations correlated with free protein S antigen levels ( r = 0.78, P < .005) (Fig 2). HIV-infected men with a prior history of thrombosis had a mean (+-SD) functional protein S value of 42.7% ? 9.1% (range 33 to 51%, n = 3) compared with a mean of 77.7% f 30.1% (range 27% to 128%, n = 22) in HIV-infected men without a previous thrombotic episode. Control men had a mean functional protein S level of 103.4% +- 20% (range 63% to 144%, n = 20). Functional protein S values were significantly different in HIV-infected men with (P < .0001) and without ( P < .003) previous thrombosis compared with control men. Free and total protein S antigen levels were also evaluated 3 to 6 months after the initial low- free protein S determination in 13 HIV-infected males. No significant differences between initial and follow-up protein S determinations for the group were seen.

To determine whether the decreases in free protein S could be attributed to shifts in protein S to the complexed form, C4bBP was measured. C4bBP levels did not correlate with free protein S levels ( r = .12, NS). None of the group of HIV- infected men with decreased free protein S had increases in C4bBP. Two HIV-infected men who had normal levels of free protein S had increased C4bBP (Fig 3). Similar results were obtained with both the Laurell electroimmunoassay and the Liatest C4bBP. To obtain a qualitative estimate of the distribution of bound and free protein S, crossed immunoelectrophoresis was performed. A reduction in the height of the lower, more rapidly migrating free protein S peak was observed in 18 of 25 study patients examined (Fig 4), con- firming the decreased levels of free protein S obtained on the Laurell procedure. However, no changes were seen in the pattern of distribution of free and bound protein S. Variant protein S molecules have been shown in familial protein S deficiency.21 Immunoblot studies were performed on the 25 study patients to exclude the presence of a variant protein S molecule. Twenty-four patients had normal protein S im- munoblots. One patient (patient 9, Table l), without a previous history of thrombosis, showed both a variant and normal protein S molecule, with the variant protein S antigen migrating abnormally fast at approximately 66,000 molecular weight (mol wt) compared with the normal protein S at approximately 7 1,000 mol wt.

The protein S status ofthe study group was compared with HIV disease seventy, degree of immunodeficiency, and medications. No correlation was seen between protein S deficiency and CDC classification, CD4+ cell levels, p24 antigen status, AZT use, or Pneumocystis carinii prophylaxis. There was a statistically significant correlation between decreases in protein S and duration of HIV disease ( r = .37, P < .05).

IgG and IgM anticardiolipin antibodies were correlated with the free protein S levels (Fig 5). A significant linear re-

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U C

._ c 2 90

.E 80

n 70 4 J

60

t 0

t

4 ., u.

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HIV with S Def HIV with nl S Control Males

Fig 3. C4b binding protein (C4bBP) in 25 HIV-study patients compared with 21 controls (Nl). HIV study patients were further divided by protein S status. The three patients with previous thrombosis are indicated by (0). Two HIV patients with normal protein S levels had increased C4bBP. No statistically significant differences existed between the groups. Data shown here were performed by the Laurell assay.




HIV AND PROTEIN S DEFICIENCY 1805

Fig 4. Crossed immunoelectrophoresis of protein S in normal plasma (A) and in two HI;' study patients (B and C). The two immunoprecipitation peaks associated with protein S are shown. The taller, slower-mi- gratingpeakrepresentsthebound protein S, while the lower, faster- migrating peak represents free protein S. Eighteen of 25 HIV- infected patients had a pattern similar to (C) with lower free protein S peaks than those observed in normal plasma. A

lationship existed between increasing IgG anticardiolipin antibody levels and decreasing free protein S levels (r = .67, P < .005). vWFAG, in contrast, did not correlate with free protein S levels (r = .I3, NS).

DISCUSSION

Protein S deficiency was commonly found in HIV-infected men. In our study, the incidence of decreased free protein S below the lower level established for the control male population was higher (76%) than that reported by others (31%).15 One possible explanation for this discrepancy may be the longer mean duration of HIV infection in our study group compared with others. Although stage of disease, CD4 counts, and p24 antigen positivity did not correlate with decreased levels of free protein S, the duration of disease did statistically correlate with decreases in protein S. A similar lack of correlation with more advanced disease has been noted in one study,I5 but not in another."

C 8 0)

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g7

1

t " 00 I

0 ' ' ' ' ' ' ' ' 1 1 ' ' 1 '11 10 20 30 40 50 W 70 80 90 103 110 120 130 " 560

IgG Anticardiolipin Antibody (GPL units)

Fig 5. Correlation between free protein S antigen levels and lgG anticardiolipin antibody levels in 24 study patients. A linear regression line is drawn. A significant correlation is seen between increasing IgG-anticardiolipin-antibody levels and decreasing free protein S antigen levels (r = .67. P < .005). lgG anticardiolipin antibody levels are expressed in standard GPL units where 1 unit is equivalent to 1 rg/mL of an affinity-purified lgG standard sample (see Materials and Methods).

=-+ B C

+

The occurrence of thromboembolic events in 3 of 25 randomly selected long-term HIV-infected men, as well as the associated findings of free protein S deficiency, elevated vWF AG, and increased IgG-anticardiolipin antibodies in all 3 patients with a history of thromboembolic disease, appears significant. Elevations in vWFAG have been associated with a large number of chronic diseases3* including HIV disease.33." Although it has been associated with conditions in which change or injury to the endothelium occurs, including ex- perimental'venous occl~sion.'~ it has not been shown to predispose to clinical thrombosis. Increases in anticardiolipin antibodies, although associated with thrombosis in systemic lupus erythematosus?' have not been linked with thrombosis in HIV di~ease.~'-~" Based on these data, reduced protein S levels appear the most likely to contribute to the risk for thrombotic disease in HIV-infected men. It remains unclear whether increasing longevity in this population poses a constant increase in thrombotic risk. Of note, in contradistinction to this hypothesis, is the occurrence of thrombosis within I year of the onset of HIV symptomatology in two of the individuals in this study. Cross-sectional or longitudinal studies are needed to clarify this.

Because C4bBP is an acute-phase reactant protein, elevations of C4bBP at times of inflammation can shift the protein S from the free to the bound form. This shift results in a decrease in free protein S antigen and in protein S functional activity. However, protein S deficiency secondary to HIV disease results in decreased free and total protein S antigen levels, but normal C4bBP levels, suggesting these changes may be different than occur in an inflammatory response. Similar findings of normal C4bBP associated with reduced total protein S and quantitatively greater reduced free protein S levels have been associated with oral contraceptive use? pregnancy: and liver disease." This relatively greater decrease in free protein S is as expected when the relationship between free protein S level, total protein S antigen, and C4bBP is calculated from the dissociation constant of the protein S/ C4bBP complex.' In systemic lupus erythematosus, reduced free and normal total protein S have been rep~r ted , '~ suggesting a shift in distribution to the bound form caused by an increase in C4bBP levels.

The pathogenesis of the decrease in protein S levels associated with HIV disease remains to be elucidated. Protein S




1806 STAHL ET AL

is synthesized by e n d ~ t h e l i u m , ~ ~ . ~ ’ hepatocyte^,'^ and mega- k a r y o c y t e ~ . ~ ~ In view of the lack of decrease in protein C, fibrinogen, and factors I1 and X, the current study does not implicate impaired hepatic synthesis of protein S as a possible cause for the HIV-associated decrease in free protein S. Be- cause abnormalities in several other substances secreted by the endothelium and involved in coagulation have been associated with HIV infection,34 disturbances in endothelial function may play a role in the etiology of the reduced protein S. As has been observed by others,33 the present study showed that vWF was increased in association with HIV disease, although no correlation was found between increases in vWF and decreases in protein S. Other mechanisms besides altered production or secretion may occur with HIV infection, re- sulting in the observed findings. The linear correlation between increased IgG-anticardiolipin antibodies and reduced free protein S levels seems relevant in this regard. Antiphos- pholipid antibodies have been postulated to interfere with the activated protein C/protein S complex at the phospholipid surface4’ and inhibition of the activated protein C complex has been ~hown.~’ .~ ’ A n abnormal interaction of protein S with the phospholipid membrane has also been postulated in association with antiphospholipid antibodies and functional protein S def i c i en~y .~ ’ ,~~ Other reports have also linked antigenic protein S deficiency with the presence of lupus an- t i c o a g u l a n t ~ , ~ ~ . ~ ~ suggesting the possibility that protein S, in addition to its affinity for phospholipid membranes, binds to antiphospholipid antibodies. In fact, anticardiolipin antibodies have been shown to be polyspecific and to strongly cross-react with other anionic phospholipid^.^^

Taken together, the results suggest that HIV-associated protein S deficiency may predispose infected individuals to thrombosis. The full clinical implications of these findings remain to be ascertained as the average life-span of HIV- infected patients increases with more effective antiviral agents and treatment of opportunistic infections. This study provides a strong rationale for further studies at the cellular level to elucidate the etiology of the decrease in protein S and the data strongly suggest that the presence of anticardiolipin or related antibodies may play a role in its pathogenesis.

ACKNOWLEDGMENT

The authors thank Tim Green for statistical assistance and Martha Byrd, Tamara Hine, and Danny Jue for technical assistance with anticardiolipin-antibody levels and amino acid determinations. Moreover, the authors thank Dr John Griffin for helpful discussions.

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3. Comp PC, Esmon C T Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 3 1 1: 1525, 1984

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14. Keeling DM, Campbell SJ, Mackie IJ, Machin SJ, Isenberg DA: Total and free protein S in Fystemic lupus erythematosus. Thromb Res 60:237, 1990

15. Lafeuillade A, Alessi MC, Poizot-Martin I, Dhiver C, Quilichini R, Aubert L, Gastaut JA, Juhan-Vague I: Protein S deficiency and HIV infection. N Engl J Med 324:1220, 1991 (letter)

16. Comp PC: Hereditary disorders predisposing to thrombosis, in Coller B (ed): Progress in Hemostasis and Thrombosis, vol 8. Or- lando, FL, Grune & Stratton, 1986, p 71

17. Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI: Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med 310:559, 1984

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1993 81: 1801-1807

CP Stahl, CS Wideman, TJ Spira, EC Haff, GJ Hixon and BL Evatt virus infection [see comments]Protein S deficiency in men with long-term human immunodeficiency

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