Skip Navigation

Association of Hypercoagulable States and Increased Platelet Adhesion and Aggregation with Bacterial Colonization of Intravenous Catheters

  1. Daniel Musher1,2,
  2. Elizabeth Goldsmith1,
  3. Sherry Dunbar1,
  4. Gordon Tilney1,
  5. Rabih Darouiche1,2,
  6. Qinghua Yu2,
  7. José A. López1,2 and
  8. Jing-fei Dong1,2
  1. 1Medical Service, Veterans Affairs Medical Center, and
  2. 2Department of Medicine, Baylor College of Medicine, Houston, Texas
  1. Reprints or correspondence: Dr. Daniel Musher, Infectious Disease Section, Veterans Affairs Medical Center, Houston, TX 77030 (daniel.musher{at}med.va.gov)
 
Next Section

Abstract

Bacterial adherence to intravenous catheters may be mediated, in part, by adherence to coagulation proteins and platelets. The possibility that catheter infection is associated with gene polymorphisms that cause hypercoagulability or increased platelet stickiness was examined. Among patients with infected catheters, there was no increase in the frequency of polymorphisms that increase coagulability, including factor V Leiden R506G, factor II (prothrombin) G20210A, and methylenetetrahydrofolate reductase C677T, compared with control subjects. The incidence of polymorphisms of the platelet β3 integrin among patients with infected catheters was also similar to that among control subjects. The C/D heterozygote of the variable number tandem repeat polymorphism and the C/T heterozygote of the KO polymorphism of glycoprotein Ibα were more frequent among patients with infected catheters than they were among control subjects. In a small proportion of patients, a genetic predisposition to platelet stickiness may be associated with infection of intravenous catheters, but in the majority, a recognized genetic tendency to hypercoagulability or platelet stickiness does not underlie infection

Infection at the site of an intravascular device is a major source of morbidity; these infections occur frequently and add substantially to the duration and cost of hospitalization [1, 2]. The first step in the evolution of such infections is the adherence of bacteria to the catheter or the adjacent vascular endothelium. Although direct adherence to plastic may occur, depending on the species of bacterium, such adherence is thought to be greatly enhanced by the prior deposition of platelets or plasma proteins that interact with fimbriae, lipoteichoic acid, and other terminal structures on bacterial surfaces [3]

In recent years, certain gene polymorphisms have been found to predispose to a hypercoagulable state [4, 5]; included are those that encode factor V Leiden R506G, factor II (prothrombin) G20210A, or methylenetetrahydrofolate reductase (MTHFR) C677T (table 1). In addition, polymorphisms of the platelet β3 integrin (PLA), which associates with the αII integrin to form the platelet receptor for fibrinogen [6–9], or of GPIbα (variable number tandem repeat [VNTR], KO, or Kozak), the receptor for von Willebrand factor, thrombin, and P-selectin [10–13], might affect the stickiness of platelets, thereby predisposing to thrombosis. Both glycoproteins are critical in primary hemostasis and arterial thrombosis, mediating platelet adhesion to subendothelium and platelet aggregation [14, 15]. The objective of this study was to determine whether there is an association between polymorphisms that result in a hypercoagulable state or in increased adhesion or aggregation of platelets and the development of infection at the site of an intravenous catheter; the polymorphisms that we studied are listed in table 1. Because deposition of fibrin or a fibrin-platelet complex is believed to be the initial pathophysiologic event in bacterial endocarditis, we also studied a small number of patients with this disease

Figure 1
View larger version:
Figure 1

Distribution of variable number tandem repeat polymorphisms of GPIbα among patients with infected catheters and control subjects. The difference between the groups was significant only for C/D heterozygosity

Figure 2
View larger version:
Figure 2

Percentage of patients with repeated catheter infections and total no. of such infections in this study of the association between gene polymorphisms implicated in hypercoagulable states and infection of intravenous catheters

Table 1
View larger version:
Table 1

Polymorphisms associated with hypercoagulable states

Previous SectionNext Section

Subjects and Methods

SubjectsSeventy-five consecutive patients were selected for study for whom culture of the terminal 2–4 cm of a removed intravenous catheter yielded >15 cfu of bacteria. Of these catheters, 67 ( 89%) yielded >100 cfu, 5 (7%) yielded 50–99 cfu, and 3 (4%) yielded 15–49 cfu. Herein, these will be referred to as “infected catheters.” Control subjects, 1–2 for each patient (n=130), were selected at random from among persons who were in the hospital at the time each patient was identified. We assumed that these control subjects reflected the universe of persons from which our medical center draws its population. Thus, the genetic composition of patients with condition “x” can be compared with the population at large, provided that a sample of appropriate size is obtained at random. A tube of uncoagulated blood from each patient was obtained in the Clinical Hematology Laboratory (Veterans Affairs Medical Center, Houston); a tube from 1 or 2 additional subjects was also taken at random from among those submitted the same day. Twelve patients who had bacterial endocarditis (positive blood culture results and documentation of vegetations by transesophageal echocardiography) were also identified during the time this study was in progress, and a discarded tube of uncoagulated blood was obtained for each of them. Names of subjects were removed before samples were sent for genotyping

Gene polymorphisms for factor V Leiden R506G, factor II G20210A, and MTHFR C677TGenomic DNA from buffy coat specimens was extracted (Generation Capture Plate Kit; Gentra Systems), amplified by polymerase chain reaction, and detected by use of the LabMAP system (Luminex). In this system, polystyrene microspheres are internally dyed with 2 distinct fluorochromes to create an array of microsphere sets with specific spectral addresses. A 6-member multiplexed assay was constructed by covalently coupling specific oligonucleotide capture probes to distinct microsphere sets [16, 17]. Coupled microsphere sets were added to denatured, amplified genomic DNA and hybridized at 52°C for 15 min. Hybridized targets were detected by use of a streptavidin-fluorophore reporter and a Luminex 100 analyzer. Fluorescence associated with each microsphere set was quantified and used to identify normal and mutant DNA sequences present in the amplified specimen. Net median fluorescence intensity for known control samples was used to establish cutoff points for normal, heterozygote, and homozygous DNA sequences

Gene polymorphisms for platelet glycoproteinsGenomic DNA was extracted from whole blood with use of a Qiagen DNA extraction kit. DNA fragments spanning the polymorphic sites were amplified by polymerase chain reaction and digested with specific restriction enzymes that generated unique digestion patterns for each genotype by 2% agarose gel electrophoresis [6, 12, 18]. This method was based on previous studies showing that the PLA2 allele of the β3 integrin and the Met 145 and −5C alleles of GPIbα create unique restriction sites for MspI, SfaNI, and PpuMI, respectively [6, 12, 19]. All enzymes were purchased from New England Biolabs. The VNTR polymorphism of GPIbα, which is characterized by various numbers of 39-bp repeats, was directly determined on the basis of the sizes of the polymerase chain reaction DNA fragments on agarose gels [18]

Statistical analysisThe number of patients reported in each comparison varies, because DNA amplification was not uniformly successful. The distribution of the gene polymorphisms among patients with infected catheters or endocarditis was compared, using an odds ratio, with that among control subjects; 1- or 2-tailed P values were calculated, and 95% confidence levels were determined. The Mann-Whitney U test was used to determine whether a specific gene allele of platelet adhesion receptors was overly represented in either the control subject or the patient population

Previous SectionNext Section

Results

Coagulation factorsAs shown in table 2, the prevalence of factor V Leiden R506G and factor II G20210A was similar among control subjects and patients with either infected catheters or endocarditis. The prevalence of MTHFR C677T, a polymorphism associated with a hypercoagulable state, was actually lower among patients with infected catheters than among control subjects (P<.05)

Table 2
View larger version:
Table 2

Prevalence of genetic factors predisposing to hypercoagulable states among patients with infected catheters or endocarditis and control subjects

Platelet surface glycoproteinsAs shown in table 3, the PLA2 polymorphism of the β3 integrin, which has previously been found to result in increased platelet adhesion and aggregation, was present in approximately the same proportion among patients with infected catheters or endocarditis and control subjects (for patients with infected catheters vs. control subjects, U=1.013 and P>.05, by the Mann-Whitney U test). For the VNTR polymorphism of GPIbα, we found no difference in the prevalences of B/C and C/C, the most common genotypes, when we compared patients with infected catheters and control subjects (figure 1). However, the C/D heterozygote was detected in a significantly greater proportion among patients with infected catheters (11.1%) than among control subjects (5.4%; U=2.341 and P<.05, by the Mann-Whitney U test). The frequency of the KO polymorphism of GPIbα among control subjects was similar to that among patients with either infected catheters or endocarditis (table 3; U=1.114 and P>.05, by the Mann-Whitney U test). As shown in table 3, the C/T heterozygote of the Kozak genotypes of platelet GPIbα was significantly more prevalent among patients than among control subjects (U=2.447 and P<.05, by the Mann-Whitney U test)

Table 3
View larger version:
Table 3

Distribution of PLA genotypes of platelet β3 integrin among patients with infected catheters or endocarditis and control subjects

Patients with multiple catheter infectionsOnce a patient was found to have an infected catheter, we searched the medical records for the preceding 2 years for earlier evidence of a catheter infection. Using this approach, we discovered that some subjects had experienced repeated bouts of catheter-associated infection. Of the 75 subjects studied, 38 (51%) had had infected catheters on ⩾2 occasions (figure 2). The greatest number of infected intravenous catheters in a single patient was 6 (concurrent infection of ⩾2 catheters was not found in any patient); this occurred in 3 patients during the 2-year period

Association between individual bacterial species and polymorphismsBecause certain organisms, such as Staphylococcus aureus are thought to attach more readily to catheters by association with human proteins [3], we examined the possibility that isolation of this organism from a catheter was associated with a polymorphism that causes a hypercoagulable state. Within the limitations of sample size, in 26 cases involving S. aureus there was no apparent association

Previous SectionNext Section

Discussion

Infection and thrombosis are the 2 most common serious complications of intravascular devices [3]. It is generally perceived that an association exists between the infectious and thrombotic complications associated with central venous catheters [20]. Postmortem studies have shown an association between mural thrombi in catheterized veins and the premortem occurrence of catheter-related septicemia [21], and prospective clinical trials have found that bonding of heparin to the surface of central venous catheters is associated with lower rates of catheter-related infection and venous thrombosis [22, 23]. It is not known whether this association reflects a causal relationship and, if so, which of the 2 complications occurs first. Earlier studies clearly showed that the fibrin sheath that engulfs indwelling vascular catheters promotes the adherence of some bacteria, particularly staphylococci [24, 25]. Bacteria adhere to mammalian proteins, especially fibrin, thrombin, and fibronectin, and to platelets, all of which deposit on the surfaces of indwelling venous catheters [3]. Clotted blood may also serve as an enriching environment for bacterial growth

In recent years, certain gene polymorphisms have been found to predispose to a hypercoagulable state [4, 5]; included are those that encode factor V Leiden R506G, prothrombin G20210A, and MTHFR C677T. In addition, polymorphisms of platelet adhesion receptors, such as PLA of the platelet β3 integrin and GPIbα (VNTR, KO, or Kozak), may also affect platelet reactivity, thereby contributing to the development of thrombosis. Persons with rare alleles of these gene polymorphisms, including the PLA2 genotype of the β3 integrin and the C/T genotype of the GPIbα Kozak polymorphism, the platelet receptor for von Willebrand factor, are at increased risk for thrombosis [26, 27]. It seemed reasonable, therefore, to investigate the prevalence of polymorphisms that are clearly associated with hypercoagulable states in patients who had bacterial deposition at the terminal part of an indwelling intravenous catheter

The results of our study show that, among patients with infected catheters and those with bacterial endocarditis, there is no increased prevalence of alleles for factor V Leiden, prothrombin G20210A, or the thermolabile variant of MTHFR that are known to be associated with hypercoagulability, although the small number of patients with endocarditis in our study precludes conclusive analysis. The PLA genotypes of the platelet β3 integrin and the genotypes of the GPIbα KO polymorphism were present with equal frequency among patients and control subjects. In contrast, the C/T heterozygote of the GPIbα Kozak polymorphism was more prevalent among patients with infected catheters. This is consistent with previous studies that showed that presence of the C allele results in a higher risk of thrombosis [27], suggesting that the prothrombotic C allele of GPIbα Kozak polymorphism may contribute to the development of microthrombi at the end of central venous catheters. This result also provides further evidence linking a thrombotic event to infection. We also found that the C/D heterozygote of the GPIbα VNTR polymorphism was overly represented in the patient population. Whether a correlation exists between the VNTR polymorphism of GPIbα and thrombosis has been controversial [19, 28, 29]; our results support the possibility that they are associated. An obvious association with any of the polymorphisms we studied was not detected among patients with endocarditis; however, the number of patients in that group was small. It is worthwhile to note that the prevalence of these various polymorphisms among our control patients was quite similar to that reported for the US population at large [13]

In conclusion, we found a higher prevalence of the C/D heterozygote genotype of GPIbα VNTR and the C/T heterozygote genotype of platelet GPIbα Kozak among patients who had infected intravenous catheters than among control subjects. We did not find any association between infection at a catheter site and other genotypes that are known to increase platelet adhesion and aggregation or those that cause a hypercoagulable state. We also found that some patients who experience infection at a catheter site have a tendency to develop repeated infections; to our knowledge, this predisposition has not been fully appreciated, and it supports the need to continue the search for host factors associated with catheter infection. Future molecular studies should examine the role of host factors, including tissue ligands such as fibronectin, fibrinogen, and fibrin, as well as bacterial factors, in the predisposition to develop such infections

Previous SectionNext Section

Footnotes

  • ↵This work was carried out under a protocol approved by the Institutional Review Board, Baylor College of Medicine

    Financial support: Department of Veterans Affairs, American Heart Association, Texas Affiliate (Merit Review funding); National Institutes of Health (grant P50-HL65967)

  • Received December 21, 2001.
  • Revision received March 20, 2002.
Previous Section
 

References

  1. ↵
    1. Pittet D,
    2. Tarara D,
    3. Wenzel RP
    . Nosocomial bloodstream infection in critically ill patients: excess length of stay, extra costs, and attributable mortality. JAMA 1994;271:1598-601.
    Abstract/FREE Full Text
  2. ↵
    1. Culver DH,
    2. Gaines RP
    . Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. Crit Care Med 1999;27:887-92.
    CrossRefMedlineWeb of Science
  3. ↵
    1. Darouiche RO
    . Device-associated infections: a macroproblem that starts with microadherence. Clin Infect Dis 2001;33:1567-72.
    CrossRefMedlineWeb of Science
  4. ↵
    1. Lane DA,
    2. Grant PJ
    . Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood 2000;95:1517-32.
    FREE Full Text
  5. ↵
    1. Seligsohn U,
    2. Lubetsky A
    . Genetic susceptibility to venous thrombosis. N Engl J Med 2001;344:1222-31.
    CrossRefMedlineWeb of Science
  6. ↵
    1. Newman PJ,
    2. Derbes RS,
    3. Aster RH
    . The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. J Clin Invest 1989;83:1778-81.
    MedlineWeb of Science
  7. ↵
    1. Weiss EJ,
    2. Bray PF,
    3. Tayback M,
    4. et al
    . A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med 1996;334:1090-4.
    CrossRefMedlineWeb of Science
  8. ↵
    1. Csaszar A,
    2. Abel T
    . Receptor polymorphisms and diseases. Eur J Pharmacol 2001;414:9-22.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Ardissino D,
    2. Mannucci PM,
    3. Merlini PA,
    4. et al
    . Prothrombotic genetic risk factors in young survivors of myocardial infarction. Blood 1999;94:46-51.
    Abstract/FREE Full Text
  10. ↵
    1. López JA
    . The platelet glycoprotein Ib-IX complex. Blood Coagul Fibrinolysis 1994;5:97-119.
    MedlineWeb of Science
  11. ↵
    1. Gonzalez-Conejero R,
    2. Lozano ML,
    3. Rivera J,
    4. et al
    . Polymorphisms of platelet membrane glycoprotein Ib associated with arterial thrombotic disease. Blood 1998;92:2771-6.
    Abstract/FREE Full Text
  12. ↵
    1. Afshar-Kharghan V,
    2. Li CQ,
    3. Khoshnevis-Asl M,
    4. López JA
    . Kozak sequence polymorphism of the glycoprotein (GP) Ibα gene is a major determinant of the plasma membrane levels of the platelet GP Ib-IX-V complex. Blood 1999;94:186-91.
    Abstract/FREE Full Text
  13. ↵
    1. Lane DA,
    2. Grant PJ
    . Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood 2000;95:1517-32.
    FREE Full Text
  14. ↵
    1. López JA,
    2. Dong JF
    . Structure and function of the glycoprotein Ib-IX-V complex. Curr Opin Hematol 1997;4:323-9.
    Medline
  15. ↵
    1. Bennett JS
    . Platelet-fibrinogen interactions. Ann NY Acad Sci 2001;936:340-54.
    MedlineWeb of Science
  16. ↵
    1. Fulton R,
    2. McDade R,
    3. Smith P,
    4. Kienker L,
    5. Kettman J
    . Advanced multiplexed analysis with the FlowMetrix system. Clin Chem 1997;43:1749-56.
    Abstract/FREE Full Text
  17. ↵
    1. Dunbar SA,
    2. Jacobson JW
    . Application of the Luminex LabMAP in rapid screening for mutations in the cystic fibrosis transmembrane conductance regulator gene: a pilot study. Clin Chem 2000;46:1498-500.
    FREE Full Text
  18. ↵
    1. López JA,
    2. Ludwig EH,
    3. McCarthy BJ
    . Polymorphism of human glycoprotein Ibα results from a variable number of tandem repeats of a 13–amino acid sequence in the mucin-like macroglycopeptide region: structure/function implications. J Biol Chem 1992;267:10055-61.
    Abstract/FREE Full Text
  19. ↵
    1. Ishida F,
    2. Ito T,
    3. Takei M,
    4. Shimodaira S,
    5. Kitano K
    . Genetic linkage of Kozak sequence polymorphism of the platelet glycoprotein Ibα with human platelet antigen–2 and variable number of tandem repeats polymorphism, and its relationship with coronary artery disease. Br J Haematol 2000;111:1247-9.
    CrossRefMedlineWeb of Science
  20. ↵
    1. Raad II,
    2. Bodey GP
    . Infectious complications of indwelling vascular catheters. Clin Infect Dis 1992;15:197-210.
    FREE Full Text
  21. ↵
    1. Raad II,
    2. Luna M,
    3. Khalil SAM,
    4. Costerton JW,
    5. Lam C
    . The relationship between the thrombotic and infectious complications of central venous catheters. JAMA 1994;271:1014-6.
    Abstract/FREE Full Text
  22. ↵
    1. Krafte-Jacobs B,
    2. Sivit CJ,
    3. Mejia R,
    4. Pollack MM
    . Catheter-related thrombosis in critically ill children: comparison of catheters with and without heparin bonding. J Pediatr 1995;126:50-4.
    CrossRefMedlineWeb of Science
  23. ↵
    1. Appelgren P,
    2. Ransjö U,
    3. Bindslev L,
    4. Espersen F,
    5. Larm O
    . Surface heparinization of central venous catheters reduces microbial colonization in vitro and in vivo: results from a prospective, randomized trial. Crit Care Med 1996;24:1482-9.
    CrossRefMedlineWeb of Science
  24. ↵
    1. Vaudaux PE,
    2. Pittet D,
    3. Haeberli A,
    4. et al
    . Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen or fibrin. J Infect Dis 1989;160:865-75.
    Abstract/FREE Full Text
  25. ↵
    1. Hermann M,
    2. Vaudaux PE,
    3. Pittet D,
    4. et al
    . Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988;158:693-701.
    Abstract/FREE Full Text
  26. ↵
    1. Bray PF
    . Integrin polymorphisms as risk factors for thrombosis. Thromb Haemost 1999;82:337-44.
    MedlineWeb of Science
  27. ↵
    1. Baker RI,
    2. Eikelboom J,
    3. Lofthouse E,
    4. et al
    . Platelet glycoprotein Ibα Kozak polymorphism is associated with an increased risk of ischemic stroke. Blood 2001;98:36-40.
    Abstract/FREE Full Text
  28. ↵
    1. Ito T,
    2. Ishida F,
    3. Shimodaira S,
    4. Kitano K
    . Polymorphisms of platelet membrane glycoprotein Ibα and plasma von Willebrand factor antigen in coronary artery disease. Int J Hematol 1999;70:47-51.
    MedlineWeb of Science
  29. ↵
    1. Mikkelsson J,
    2. Perola M,
    3. Penttila A,
    4. Karhunen PJ
    . Platelet glycoprotein IbαHPA-2 Met/VNTR B haplotype as a genetic predictor of myocardial infarction and sudden cardiac death. Circulation 2001;104:876-80.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2002) 186 (6): 769-773. doi: 10.1086/342882
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most