Platelet adhesion to collagen in healthy volunteers is influenced by variation of both α₂β₁ density and von Willebrand factor

Hemostasis and thrombosis are initiated by the adhesion of platelets to collagen.1 First, platelet velocity is reduced by the interaction between GPIb on the platelet surface and von Willebrand factor (vWF), which is attached to collagen.2,3 This allows the binding of α₂β₁ to collagen, which is necessary for firm attachment4,5; and platelet aggregation, which is activated by the collagen–GPVI interaction.5 The availability of integrin α₂β₁ on the platelet surface may play an essential role in platelet adhesion to collagen in the vessel wall (types I, III, IV, V, and VI).6 Patients with severely reduced expression of α₂β₁ on the platelet surface present with prolonged bleeding times, chronic mucocutaneous bleeding, and reduced in vitro platelet adhesion to collagen.7 8 

The number of α₂β₁ copies on the platelet surface is highly variable among healthy individuals.8This variability may correlate with quantitative estimates of platelet adhesion to collagen types I and III under static conditions.8 Variability of α₂β₁ is primarily attributable to a C807T (single-letter amino acid code) silent mutation in the gene of the α₂ subunit.9 Recently, it has been found that patients with type I von Willebrand disease who are homozygous for this mutation have a reduced risk of severe bleeding compared with C807 homozygous patients.10 Platelet vWF and plasma vWF are also subject to physiologic variation among individuals. The strongest genetic marker of reduced vWF levels known thus far is blood group O.11 At present, little is known about the influence of physiologic variations of α₂β₁ and platelet or plasma vWF levels on platelet adhesion to collagen among normal subjects. Recent evidence suggests a relation between the α₂β₁ density on the platelet surface and platelet attachment to collagen type I in whole blood at a high shear rate (1500/s).12 

We have studied the relations between genetic and acquired variations of α₂β₁ on the platelet membrane, plasma vWF levels, and adenosine diphosphate (ADP) and adenosine triphosphate (ATP) concentrations on the one hand; and platelet adhesion to collagen under flow, aggregation of platelets in the aggregometer, and the bleeding time on the other hand in a population of 32 normal subjects.

Blood was collected from 17 male and 15 female volunteers who did not take aspirin or other platelet-function inhibitors in the preceding 10 days.

Human placental collagen types I, III, and IV (Sigma, St Louis, MO) were solubilized in 50 mmol/L acetic acid (1.4 mg/mL) and sprayed on glass coverslips (Menzel, Braunschweig, Germany) with a retouching airbrush (Badger model 100; Badger Brush, Franklin Park, IL). The surface density was 30 μg/cm², supporting optimal platelet coverage.13 The coverslips with collagen were subsequently blocked by incubation with a 1% human albumin solution (Behring, Marburg, Germany) in phosphate-buffered saline (PBS) for 30 minutes at room temperature.

Monoclonal antibody (MoAb) 6F1 and MoAb 176D7, directed against the α₂ subunit of the integrin α₂β₁, were kindly provided by Dr B. Coller14 (Mount Sinai Hospital, New York, NY) and by Dr H. R. Gralnick15 (National Institutes of Health, Bethesda, MD), respectively.

The bleeding time was determined using the Simplate IIR 59271 device (Organon Technica, Durham, NC), according to Mielke.16 After bleeding time measurement, the opposite arm was used for blood sampling.

Blood was anticoagulated with 1:10 volume of 150 U/mL Orgaran (a low-molecular-weight heparinoid [LMWH]; Organon, Oss, the Netherlands). Perfusions were routinely performed in a single-pass parallel-plate perfusion chamber with a slit height of 0.1 mm,17 corresponding to a flow rate of 260 μL/min (shear rate = 1300/s). Blood was prewarmed at 37°C for 10 minutes and was drawn by a Harvard infusion pump (pump 22, model 2400-004; Natick, MA) through 4 parallel connected perfusion chambers for 5 minutes. After each perfusion run, the 4 coverslips were removed from the perfusion chambers, rinsed with PBS, fixed in glutardialdehyde (0.5% 10 mmol/L sodium phosphate, 150 mmol/L NaCl, pH 7.4), and stained with May-Grünwald-Giemsa.18 Platelet adhesion was quantified with a light microscope (at 1000× magnification) equipped with a CCD camera (JAI-CV-235C, Copenhagen, Denmark) coupled to a Matrox frame grabber (Matrox Electronic Systems, Quebec, Canada) using Optimas 6.0 software (Optimas, Seattle, WA). Three lines perpendicular to the flow direction were evaluated: 1 line in the center of the coverslip and 2 lines, 3 mm to the right and 3 mm to the left of the center. Platelet adhesion was expressed as the percentage of the surface covered with platelets.

Fixed platelets were prepared by collecting 1 volume of blood in 5 volumes of paraformaldehyde in PBS (final concentration 1%). Platelets were washed twice with PBS to which 5 mmol/L EDTA had been added (PBS/EDTA), and platelets were diluted to a concentration of 3 × 10⁸/mL. Ten microliters of platelet suspension was incubated with 5 μL biotinylated MoAb 6F1 or 5 μL biotinylated MoAb 176D7 (5 μg/mL), followed by a second incubation step with phycoerythrin-conjugated streptavidin (Becton Dickinson, San Jose, CA). After washing, the platelets were resuspended in 2 mL PBS. Platelets were analyzed in a FACScan flow cytometer at a wavelength of 488 nm.19 FACScan data were analyzed with PCLYSIS software (Becton Dickinson). As a control, biotinylated control IgG against an antigen that is not present on platelets was used. The density of α₂β₁ on the platelet surface was quantified as mean fluorescence intensity (MIF).

Whole genome DNA was isolated from 10-mL citrated blood fractions. A fragment containing nucleotide 807 located in the gene coding for the α₂ subunit of α₂β₁ was amplified in 20 mmol/L Tris/HCl, pH 8.0, 2.5 mmol/L MgCl₂, 50 mmol/L KCl, 0.1 mg/mL bovine serum albumin, 0.4 pmol of 3′ primer (5′-TGTTTAACTTGAACACATATAAAACC-3′), 0.4 pmol of 5′ primer (5′-GATTTAACTTTCCCAGCTGCCTTC-3′), 0.42 mmol/L of each nucleotide (Pharmacia Biotech, Uppsala, Sweden), 0.075 U superTAQ polymerase (HT Biotechnology Ltd., Cambridge, UK), and 5 μL DNA. Amplification was performed with an MJ Research PTC200 multicycler (MJ Research, Watertown, MA). Temperature cycles were: 4 minutes at 94°C, 33 cycles of 40 seconds at 94°C, 40 seconds at 55°C, and 2 minutes at 72 °C. The reaction was terminated with 10 minutes of incubation at 72°C. Genotype was determined from each DNA fraction by dot blotting and hybridization with antigen-specific oligonucleotides.20 The antigen-specific oligonucleotide for the C807 allele was 5′-γ³²P- AATTGCTCCGAATGTGTT-3′ and for the 807T allele was 5′-γ³²P-AATTGCTCCAAATGTGTT-3′. Dots were visualized on x-ray films (DuPont, Brussels, Belgium).

Blood was anticoagulated with 1:10 volume of 150 U/mL Orgaran or with 130 mmol/L trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation, and the platelet count was adjusted to 250 × 10³ platelets/μL. A total of 500 μL of PRP was prewarmed at 37°C for 3 minutes, and aggregation was initiated by fibrillar collagen type I or III. Fibrillar collagen types I and III were solubilized in 0.1 mol/L acetic acid at 1 mg/mL and were dialyzed twice for 24 hours at 4°C against a sodium phosphate buffer (20 mmol/L Na₂HPO₄) at pH 7.4.2Threshold values of collagen types I and III to obtain platelet aggregation were measured in a Lumiaggregometer (Chronolog, Havertown, PA) at 37°C.

Platelet aggregation in response to arachidonic acid was determined to exclude the volunteers who had taken aspirin or other platelet-function inhibitors. Blood was anticoagulated with 1:10 volume of 250 U/mL LMWH. A total of 450 μL of PRP (250 × 10³platelets/μL) was prewarmed at 37°C for 3 minutes, and aggregation was initiated by 50 μL arachidonic acid (15 mmol/L; Biodata, Horsham, PA) in a Chronolog Lumiaggregometer at 37°C.

Blood was anticoagulated in 1:10 volume of acid–citrate–dextrose anticoagulant (85 mmol/L trisodium citrate·2H₂O, 71 mmol/L citric acid, and 111 mmol/L glucose). Platelet-poor plasma (PPP) and PRP were prepared by centrifugation. PPP was immediately frozen at −70°C for the determination of vWF in plasma. After sedimentation, the platelets were washed 3 times in washing buffer (5 mmol/L sodium citrate, 0.15 mol/L NaCl, pH 6.5; containing 5 mmol/L EDTA, 10 μmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 10 ng/mL Iloprost, pH 6.5) and then resuspended at 10⁶/μL in the same buffer. Lysis was obtained by incubating the platelet suspension with 1:40 volume of 20% Triton X-100 for 1 hour at 37°C. The insoluble fraction was precipitated by centrifugation at 10 000g for 20 minutes, and the supernatant was frozen at −70°C for the determination of vWF in platelets. The vWF antigen was measured by enzyme-linked immunosorbent assay with horseradish peroxidase–conjugated anti-vWF (Dako A/S, Glostrup, Denmark).

PRP was prepared from fresh citrated blood (130 mmol/L trisodium citrate) by centrifugation. ATP and ADP in the metabolic pool as well as in the storage pool were collected by extraction with ethanol. One milliliter of PRP was mixed with 2 mL cold EDTA-ethanol solution (1:10 volume of 0.1 mol/L EDTA, pH 7.4, and 9:10 volume of ethanol 96%) and was immediately frozen at −70°C. ATP was measured by using firefly luminescence. ADP was converted into ATP by phospho enol pyruvate–pyruvate kinase (PEP-PK) (Boehringer Mannheim, Mannheim, Germany) and was measured separately by the same method.21 

PPP was prepared by centrifugation of blood anticoagulated with unfractionated heparin (5 U/mL) and was immediately frozen at −70°C. The total magnesium concentration was measured with an Ektachem Analyzer E700 XR (Eastman Kodak, Rochester, NY); the ionized magnesium concentration was measured with an ion-selective electrode from NOVA Biomedical (Waltman, MA).

The population mean and standard error of the mean were calculated for continuous variables. Dichotomous variables were expressed as the fraction of the total population. Differences in α₂β₁ density; platelet adhesion to collagen types I, III, and IV; Simplate bleeding time; and threshold values of collagen types I and III for platelets to aggregate under static conditions according to α₂β₁ genotype were analyzed with independent-sample t-test analyses. The correlations between α₂β₁ density, plasma vWF and platelet vWF, and ADP levels and ADP/ATP ratio were calculated using linear regression analysis; as were platelet adhesion to collagen types I, III, and IV; Simplate bleeding time; and threshold values of collagen types I and III for platelets to aggregate under static conditions. Subgroup analysis was performed according to α₂β₁ density lower or greater than the median and plasma vWF lower or greater than the median. Subjects who had both α₂β₁ density and plasma vWF lower than the median were defined as the reference group. Differences between subgroups and the reference group were tested by independent-sample t-test analyses. Similarly, subgroup analysis was performed on α₂β₁ density and platelet vWF.

Our study population comprised 17 men and 15 women (Table1). The average age was 36.9 years, and 8 subjects were smokers. Platelets from 5 subjects did not respond to arachidonic acid, although none of the subjects reported that they had used aspirin.

The α₂β₁ densities on the platelet membranes for α₂β₁ C807 homozygotes, C807T heterozygotes, and one 807T homozygote are plotted in Figure1. The α₂β₁ density on the platelet membrane in α₂β₁ C807 homozygotes was 17.6 ± 1.4 (MIF); this was significantly lower than in α₂β₁ C807 T heterozygotes and one 807 TT homozygote, who had an α₂β₁ density of 33.0 ± 2.6 (MIF). Unfortunately, we had only one α₂β₁ 807T homozygote, and we therefore had to classify 807 CT heterozygotes and 807T homozygotes as one group.

Table 2 shows the relation of α₂β₁ C807T genotype to α₂β₁ density; adhesion of platelets to collagen types I, III, and IV; the simplate bleeding time; and the threshold value of collagen types I and III for platelets to aggregate in the aggregometer. The α₂β₁ 807T polymorphism is associated with increased adhesion to collagen type IV under flow conditions; C807 homozygotes had 8.9% ± 2.7% coverage on collagen type IV–coated coverslips, whereas C807T heterozygotes (including one 807T homozygote) had a mean coverage of 20.4% ± 2.8%. The C807T polymorphism was not associated with the threshold value for aggregation caused by collagen types I and III or with the Simplate bleeding time.

Comparable results were found for α₂β₁density on the platelet surface. The α₂β₁density was associated with platelet aggregation to collagen types I and IV (r² = 0.35 and 0.42, respectively; Table 3). The correlation coefficient between α₂β₁ density and platelet adhesion to collagen type III was also increased, but was not statistically significant. Platelet vWF levels were associated with increased adhesion to collagen type IV (r² = 0.34) and were negatively associated with threshold values to aggregate on collagen types I (r² = −0.30) and III (r² = −0.43). Plasma vWF was associated with increased platelet adhesion on collagen types I (r² = 0.45) and III (r² = 0.42). Adjustment for age, sex, smoking, oral contraceptive use, presence or absence of arachidonic acid–induced aggregation, magnesium concentrations, and ADP and ATP levels did not influence the relations. The major dense granule substance, ADP, was not associated with platelet adhesion to collagen; however, it was inversely associated with the Simplate bleeding time (r² = −0.38). This correlation was largely attributable to extreme values in one subject; when this subject was omitted from the analysis, the relation disappeared.

The interaction between α₂β₁ density and plasma vWF on platelet adhesion to collagen types I, III, and IV is presented in Table 4. In addition, the interaction between α₂β₁ density and platelet vWF on platelet adhesion to collagen types I, III, and IV is presented in Table 4. Subjects who had both α₂β₁ density values lower than the median and plasma vWF levels lower than the median had the lowest platelet coverage on the surface of collagen types I and III (17.1% and 21.1%, respectively), followed by subjects with α₂β₁ density values greater than the median and plasma vWF lower than the median (28.8% and 28.2%, respectively) and by subjects with α₂β₁ density values lower than the median and plasma vWF greater than the median (33.4% and 30.4%, respectively). Subjects who had both α₂β₁ density values greater than the median and plasma vWF levels greater than the median had the highest platelet coverage on the surface of collagen types I and III (38.0% and 30.4%, respectively). The platelet coverage on collagen type IV was significantly increased only when subjects had both α₂β₁ density values greater than the median and plasma vWF levels greater than the median.

Subjects with both platelet vWF levels and α₂β₁ density on the platelet surface greater than the median had a 4.5-fold greater platelet aggregation on the collagen type IV surface than subjects who had both platelet vWF levels and α₂β₁ density lower than the median. Subjects with either a platelet vWF level or α₂β₁ density higher than the median had intermediate platelet adhesion to collagen. Although much weaker, a comparable relation was observed for the platelet adhesion to collagen types I and III.

In this report, we have shown that the C807T polymorphism in α₂β₁, variations in α₂β₁ density on the platelet membrane, and variations in plasma levels and platelet levels of vWF contribute to platelet thrombus formation on the collagen surface under flow.

To appreciate our findings, some characteristics of our study need to be addressed. Collagen-adhesion experiments were performed under flow conditions at a shear rate of 1300/s, which approximates the physiologic shear rate in arterioles. This allows the platelets to aggregate via the multistep process starting with the binding of vWF to collagen. Subsequently, GPIb on the platelet surface can bind vWF to reduce the velocity of platelets4,5 and to realize firm attachment of platelets to collagen through the interaction of α₂β₁ and collagen. Subsequently, platelets become activated by the collagen–GPVI interaction and aggregate through both the vWF to GPIb and α_IIbβ₃ to fibrinogen and vWF interactions.5 

Our study population was based on 32 unselected laboratory employees. Platelet-adhesion experiments, α₂β₁ density measures, and platelet and plasma antigen measures of vWF were performed without prior knowledge of the C807T genotype. Assuming that the allele frequency of the 807T polymorphism in the population was 0.4, we expected to find 5 homozygous subjects. We were unfortunate to find only one 807TT homozygote (although this was within the limits of the Hardy-Weinberg equilibrium). Despite the low number of 807T homozygotes, we observed that even intermediately increased α₂β₁ density on the platelet surface, as in C807T heterozygotes, was predictive of increased platelet adhesion to collagen. We expect that the differences in platelet adhesion to collagen would have been more convincing had we had more homozygotes.

Platelet coverage on the collagen surface was measured in a standard way after 5 minutes of perfusion. Single-time-point measures of platelet deposition may give an underestimation of the platelet adhesion rate. We expect that the differences in platelet deposition between α₂β₁ C807T genotypes would have been even more convincing if real-time measurements had been used to compare platelet deposition among subjects. Similarly, we expect that the correlation coefficients between platelet adhesion to collagen and variation of α₂β₁ density on the platelet surface and platelet and plasma levels of vWF would have been higher had we used real-time measures instead of single-time-point measures.

Our findings that platelet adhesion to collagen types I and III depends on plasma levels of vWF and that this relation is strongly enhanced by increased α₂β₁ density on the platelet surface support the view that platelet adhesion to collagen is a multistep mechanism in which the velocity of the circulating platelets is reduced by GPIb interaction with vWF bound to collagen types I and III, followed by firm attachment by α₂β₁collagen binding and subsequent platelet activation and aggregate formation.4 5 From our findings, it can be concluded that variations in plasma vWF and α₂β₁ density on the platelet surface among normal subjects are predictive of the potential of their platelets to adhere to collagen types I and III.

The finding that platelet adhesion to collagen type IV was mainly dependent on α₂β₁ expression and platelet levels of vWF, and not on plasma vWF, confirmed previous findings that vWF cannot bind to the surface of collagen type IV without the presence of platelets.17 The mechanism linking platelets to collagen type IV remains to be established.

We did not observe a relation between Simplate bleeding time and α₂β₁ density, plasma vWF, or platelet vWF. The explanation for the lack of association is that the test is too insensitive to distinguish differences in normal subjects. Therefore, the standard deviation of the test is too large, and hence the 95% confidence interval is too wide to observe a significant relation between platelet function and Simplate bleeding time among healthy subjects.

The absence of a relation between ADP levels and platelet deposition to collagen may be a consequence of the small population size of our study relative to the high standard deviations of the ADP levels. Therefore, the confidence intervals were too wide to draw firm conclusions in relation to platelet deposition to collagen. The relation between ADP levels and reduced bleeding time was mainly a consequence of extreme values in one subject. We therefore cannot draw conclusions on a relation between ADP levels and bleeding time for the total population.

In summary, plasma and platelet vWF levels and α₂β₁ expression on the platelet surface are highly variable among subjects. The variability of α₂β₁ expression is strongly associated with genetic variability of the α₂β₁ C807T polymorphism. We found that variations of plasma vWF and α₂β₁ expression on the platelet surface among normal subjects are predictive of the potential of platelets to adhere on collagen types I and III, whereas variations of platelet vWF levels and α₂β₁ expression on the platelet surface play a major role in platelet adhesion to collagen type IV.

The finding that genetic and phenotypic variations of α₂β₁ and vWF are associated with platelet adhesion to collagen may have implications for future research on the relation between platelet adhesion to collagen and cardiovascular disease. Until recently, it was difficult to study the direct relation between platelet adhesion to collagen and cardiovascular disease in large populations, and it was therefore difficult to link platelet function directly to cardiovascular disease. The discovery of the α₂β₁ C807T genotype was the first step in using markers of platelet adhesion to collagen in large epidemiologic studies. Association studies on the C807T genotype and the incidence of cardiovascular disease were suggestive of a relation.22 23Our findings that measurable differences in α₂β₁ density, α₂β₁ C807T genotype, and vWF among normal subjects are associated with platelet adhesion to collagen open new opportunities to evaluate the relations between different markers of platelet adhesion to collagen and the interaction among those markers in relation to cardiovascular disease in large populations.