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Keywords:

  • bleeding time;
  • aspirin;
  • platelet glycoprotein IIIa;
  • polymorphism;
  • thrombin generation

Abstract

  1. Top of page
  2. Abstract
  3. SUBJECTS and METHODS
  4. Results and discussion
  5. References

A single nucleotide T to C transition of the gene encoding glycoprotein IIIa leads to a common diallelic polymorphism Leu-33[RIGHTWARDS ARROW]Pro (PLA1/A2). We studied the relationship between the PlA1/A2 polymorphism and platelet function in 80 healthy men, aged 20–25 years. Before aspirin ingestion, bleeding time (BT) was shorter in carriers of the PlA2 than in carriers of the PlA1/A1 allele. At 4 h after ingestion of 300 mg of aspirin, BT became prolonged, and the intergroup difference was enhanced. In seven out of 26 PLA2 allele carriers, aspirin shortened BT on average by 30 s, compared with only one among 54 subjects with the PlA1/A1 genotype. Thus, BT both at baseline and after aspirin depends on the PlA1/A2 polymorphism of glycoprotein IIIa. Carriers of the PlA2 allele appear to be more resistant to the antithrombotic action of aspirin.

The formation of occlusive platelet aggregates involves the cross-linking of activated glycoprotein IIb/IIIa receptors on adjacent platelets by fibrinogen and other macromolecular ligands. A single nucleotide transition at position 1565 in exon 2 of the gene encoding glycoprotein IIIa (GPIIIa) leads to its diallelic polymorphism (PlA1/A2 or HPA-1a/b). Allele PlA2 (Leu-33[RIGHTWARDS ARROW]Pro) is present in 20–30% of Caucasians. The PlA2 allelic variant of GPIIIa has been reported by some, although not all, authors to be an inherited risk factor for acute coronary events (for references, see Feng et al, 1999 ). We investigated whether the PlA1/A2 polymorphism of GPIIIa could affect platelet function as assessed by bleeding time (BT), its response to aspirin and thrombin generation in vivo.

SUBJECTS and METHODS

  1. Top of page
  2. Abstract
  3. SUBJECTS and METHODS
  4. Results and discussion
  5. References

Healthy male medical students, aged 20–25 years, refrained from taking any medication for 3 weeks preceding the study. On the study day, arachidonic acid at a concentration ≤ 1·200 µmol/l induced irreversible platelet aggregation in all subjects. BT measurements were performed before and 4 h after ingestion of 300 mg aspirin. Using a Simplate II device (Organon Teknika, Durham, NC, USA), two horizontal incisions were made on a lateral aspect of the forearm at venous stasis of 40 mmHg ( Szczeklik et al, 1986 ). Measurements were performed by the same investigator, who was blinded to the results of genotyping. Prothrombin fragment 1 + 2 (F1 + 2) levels in peripheral venous blood were determined using Enzygnost (Behring, Germany). Participants were genotyped by a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). The university ethical committee approved the study, and all individuals gave informed consent.

Most variables were not normally distributed and were analysed using the Wilcoxon matched pair test or chi-square test for intragroup differences. For normal distribution, the paired t-test was used. Comparisons between the groups were made using the Mann–Whitney U-test.

Results and discussion

  1. Top of page
  2. Abstract
  3. SUBJECTS and METHODS
  4. Results and discussion
  5. References

Before aspirin ingestion, BT was shorter in the carriers of the PlA2 allele than in the PlA1/A1 group. Aspirin prolonged BT in both groups and enhanced the intergroup difference ( Table I, Fig 1). In seven out of 26 PlA2 allele carriers, BT became shorter after 300 mg aspirin, on average by 30 s, compared with only one among 54 subjects with the PlA1/A1 genotype (P = 0·001).

Table I.  Basic characteristics of two groups: PIA1A1 (n = 54) and PIA1A2 + A2A2 (n = 26).
ParameterPIA1A1PIA1A2 + A2A2P-value
  1. Values are given as median (interquartile range).

  2.  IgE, immunoglobulin E; TC, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglycerides; Fbg, fibrinogen; AT III, antithrombin III; BT, bleeding time; dt, difference in bleeding time; F1 + 2, prothrombin fragments F1 + 2; dF1 + 2, difference in F1 + 2 levels before and after aspirin; NS, not significant (P > 0·05); ASA, aspirin.

Age (years)22·621·8NS
IgE (IU/ml)27·25 (14·45; 53·45)58·9 (23·6; 94·5)NS
Platelets (× 109/l) 221 (199; 242)221·5 (188; 240)NS
TC (mmol/ml)4·22 (3·89; 4·84)4·32 (3·97; 4·65)NS
LDL-C (mmol/l)2·35 (1·93; 2·7)2·29 (1·875; 2·65)NS
HDL-C (mmol/l)1·445 (1·275; 1·71)1·415 (1·335; 1·595)NS
TG (mmol/l)0·99 (0·81; 1·22)1·06 (0·93; 1·34)NS
Fbg (g/l)2·05 (1·71; 2·28)2·01 (1·8; 2·34)NS
AT III (g/l)0·271 (0·261; 0289)0·274 (0·25; 0·298)NS
BT before ASA (s)372·5 (300; 524)302·5 (263; 345)0·006
BT after ASA (s)485 (420; 540)349 (283; 435)0·00001
dt (s)116·5 (60; 175)37·5 (−10; 87)0·0002
F1 + 2 before ASA (nmol/l)0·36 (0·3; 044)0·305 (0·25; 0·35)0·02
F1 + 2 after ASA (nmol/l)0·34 (0·25; 0·43)0·35 (0·26; 0·44)NS
dF1 + 2 (nmol/l)−0·02 (−0·09; 0·05)0·04 (−0·015; 0·095)0·016
image

Figure 1. Bleeding time (BT) before and after 300 mg aspirin in healthy men divided into two groups based on the PIA1/A2 polymorphism. Left: BT before aspirin. Right: difference between BT measured before and 4 h after 300 mg aspirin. (○) carriers of the PIA1/A1 genotype; (●) carriers of the PIA2 allele.

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At baseline, lower levels of F1 + 2 were found in PlA2 carriers. After aspirin, F1 + 2 levels showed a mild tendency to decrease in subjects with the PlA1/A1 genotype and to increase in carriers of the PlA2 allele (P = 0·07), resulting in levelling-off the groups. After aspirin, F1 + 2 levels increased in 65% of men with the PlA2 allele but only in 37% of subjects positive for the PlA1/A1 genotype (RR 3·2; 95% CI 1·2–8·0; P = 0·02). There was no significant correlation between BT and plasma levels of F1 + 2 in either group both before and after aspirin administration.

The only data on BT in subjects genotyped for the PlA1/A2 polymorphism were reported by de Maat et al (1997) who found no difference between carriers and non-carriers of the PlA2 allele among Greenland Inuits using a different method for BT determination. Genetic differences and long BT in Inuits could explain these different results.

We studied BT at baseline and 4 h after aspirin administration, when the increase in BT is maximal, regardless of the dose of the drug ( Fiore et al, 1990 ). All the measurements were made by the same investigator, blinded to the results of genotyping, which enhances the reliability of our results. The two groups studied were almost identical with regard to demographic, clinical and biochemical characteristics, including the confounding factors such as smoking, cholesterol or IgE levels ( Fiore et al, 1990 ; Szczeklik et al, 1996 ; Kauhanen et al, 1998 ), known to influence BT. Therefore, the significant intergroup difference in BT at baseline, which became intensified by aspirin, may be attributed to the carrier status of the PlA2 allele. The different behaviour of thrombin generation markers in the studied groups could be result either from increased binding of other macromolecular ligands to GPIIb/IIIa receptor in carriers of PLA2 allele or from some unknown homeostatic mechanisms, compensating for the shortened BT. After aspirin administration, this difference waned. Impaired inhibition of thrombin generation by aspirin in PlA2 carriers has been observed at the site of microvascular injury ( Undas et al, 1999 ).

Substitution of proline for leucine (PLA2) is associated with an increased binding of fibrinogen to the platelet GPIIb/IIIa receptor ( Goodall et al, 1999 ); this would explain the difference in BT at baseline between carriers of the two alleles. Aspirin could directly affect regulation of GPIIb/IIIa by interfering with intracellular signalling events ( Dominguez-Jimenez et al, 1999 ). Interestingly, the effects of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) on IIb/IIIa activation appear to be exerted by cyclooxygenase-dependent ( Peter et al, 1998 ) and cyclooxygenase-independent ( Dominguez-Jimenez et al, 1999 ) action.

The findings reported here may help to explain the functional aspirin resistance in some cardiac patients ( Kottke-Marchant et al, 1999 ) and the failure of aspirin to protect against atherothrombosis and its complications in some stroke patients ( Grotemeyer et al, 1993 ). Kottke-Marchant et al (1999) reported that 6–11% of cardiac patients taking aspirin do not achieve optimal functional platelet inhibition, and an additional 23% have suboptimal inhibition. Perhaps such aspirin resistance depends, partly at least, on the PlA1/A2 polymorphism. In that case, the receptor antagonists mimicking the peptide motifs of high receptor affinity might be the best choice of antithrombotic therapy in subjects with the PlA2 allele.

References

  1. Top of page
  2. Abstract
  3. SUBJECTS and METHODS
  4. Results and discussion
  5. References
  • De Maat, M.P., Bladbjerg, E.M., Johansen, L.G., Bentzen, J., Jespersen, J. (1997) PlA1/A2 polymorphism of platelet glycoprotein IIIa and risk of cardiovascular disease [letter]. Lancet, 349, 1099 1100.
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