Marked Temperature Dependence of the Platelet Calcium Signal Induced by Human von Willebrand Factor

The dissociation constants for binding of calcium to Fura-PE3 at 20°C and 37°C were assessed using a calcium calibration buffer kit (Molecular Probes, Eugene, OR) in accordance with the manufacturer’s instructions. In brief, for measurements at each temperature, two solutions containing 2 μmol/L Fura-PE3 (K⁺ salt; Texas Fluorescence Laboratory, Austin, TX) were prepared, one with 10 mmol/L EGTA and the other with 10 mmol/L CaEGTA. Both solutions also contained 100 mmol/L KCl, 1 mmol/L free Mg²⁺, and 10 mmol/L 3-(N-morpholino) propanesulfonic acid (MOPS), and they were adjusted to pH 7.0 at the desired temperature. A series of 11 solutions of known free Ca²⁺ concentration was prepared by appropriate mixing. Fluorescence excitation spectra were collected for each solution at the appropriate temperature with an emission wavelength of 510 nm. The kd value was calculated from a Hill plot of the fluorescence intensity with excitation at 340 nm.17 

Venous blood was drawn from healthy adult volunteers who had not taken aspirin or a nonsteroidal antiinflammatory drug for at least 10 days before phlebotomy. Female donors were not taking an oral contraceptive. The protocol was approved by the local Institutional Review Board, and informed consent was obtained from each donor. Platelets were isolated using the method described previously.17 In brief, blood was collected into 1/6 volume of acid-citrate-dextrose anticoagulant (65 mmol/L citric acid, 85 mmol/L sodium citrate, 110 mmol/L D-glucose). Platelet-rich plasma was treated with 5 mmol/L creatine phosphate and 25 U/mL creatine phosphokinase (Sigma Chemicals, St Louis, MO), and the platelets were isolated by centrifugation at 1,000g for 15 minutes at 23°C. The platelets were resuspended in calcium-free, magnesium-free, Tyrode’s solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L NaHCO₃, 0.4 mmol/L NaH₂PO₄, 5.5 mmol/L D-glucose), supplemented with 10 mmol/L HEPES, pH 7.35, and 0.35% (wt/vol) bovine serum albumin (BSA). This solution was also supplemented with 5 mmol/L creatine phosphate, 25 U/mL creatine phosphokinase, and 1 mmol/L EGTA during indicator loading.

Platelets were loaded with fluorescent calcium indicator by a 1-hour incubation at 23°C in the dark with 5 μmol/L Fura-PE3 acetoxymethyl ester (AM) (Texas Fluorescence Laboratory) in the presence of 0.004% (wt/vol) Pluronic F-127; before addition to the platelets, the Fura-PE3/AM stock (3.3 mmol/L in dimethylsulfoxide) was mixed with Tyrode’s solution containing 4% BSA to facilitate its dispersal. A separate portion of the platelet suspension was retained as an autofluorescence control. After loading, the platelets were washed twice by centrifugation and resuspension.17,21 The final resuspension (in buffered Tyrode’s solution with 0.35% BSA and 1 mmol/L MgCl₂) was adjusted to a platelet count of 50,000 cells/μL.

Platelet [Ca²⁺]_i was assayed on a Photon Technology International (South Brunswick, NJ) RF-M2004 spectrofluorometer. All studies were conducted in a cylindrical glass cuvette (8-mm diameter) designed for aggregometry (Payton Scientific, Buffalo, NY). Reflections from its curved surface were eliminated by using a vertical polarizer in the excitation beam and a horizontal one in the emission beam. A specially designed stirrer was used; this comprised a Teflon cylinder with a bar magnet embedded at its base and an ultraviolet (UV)-grade methacrylate stirring vane attached at the top.17 This stirrer ensures efficient stirring of a platelet suspension without significant interference with the optical signal, and its use has been validated extensively in studies with both classical platelet agonists and multimeric human vWF.17 

Platelets (50,000 cells/μL) that had been loaded with fluorescent calcium indicator were stirred continuously at the selected temperature. They were equilibrated briefly (1 to 2 minutes) with extracellular calcium (1 mmol/L CaCl₂), unless otherwise indicated. Stimulation was usually effected by sequential addition of 1 mg/mL ristocetin (Bio/Data Corp, Hatboro, PA) and 5 μg/mL purified human vWF. Human vWF was either obtained from a commercial source (Calbiochem, San Diego, CA) or purified from plasma by cryoethanol precipitation, differential polyethylene glycol fractionation, and gel filtration on Sepharose CL-4B.22,23 Similar results were obtained with these two vWF preparations; each was essentially pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and included the full range of multimers present in plasma vWF. In other studies, platelets were stimulated by addition of 5 μmol/L adenosine 5′-diphosphate (ADP), 0.02 U/mL human α-thrombin, 0.1 μmol/L U-46619, or 20 μmol/L arachidonic acid (all from Sigma Chemicals). Fluorescence intensity was monitored at 510 nm emission with excitation alternating between 340 and 380 nm wavelengths. Autofluorescence was determined in platelets that had not been loaded with Fura-PE3 and was subtracted from each fluorescence measurement (before ratio calculation).

Calibration data were obtained in each experiment by the procedure of Merritt et al24 (on a separate aliquot of platelets). The maximal fluorescence ratio (R_max) was assessed by adding 60 μmol/L digitonin (Calbiochem) in the presence of 1 mmol/L CaCl₂, and the minimal fluorescence ratio (R_min) determined by adding excess (20 mmol/L) EGTA, buffered to pH 8.5. Experimental measurements of the fluorescence ratio (R) were converted to [Ca²⁺]_i values by the equation25:

where F_f and F_b are the fluorescence intensities with 380 nm excitation for free and calcium-bound indicator, respectively. Calculations at 20°C and 37°C are based on measured kd values of 251 and 146 nmol/L, respectively, for binding of calcium to Fura-PE3 (see Results); calculations at intermediate temperatures used interpolated kd estimates of 220 nmol/L at 25°C and 189 nmol/L at 30°C.

Platelet aggregation was assessed simultaneously with the [Ca²⁺]_i measurements in some studies. The Photon Technology RF-M2004 spectrofluorometer was used in an unorthodox configuration for these studies (Fig 1); one emission monochromator (380 nm wavelength) was repositioned to monitor transmitted light, and thereby assess aggregation, while platelet [Ca²⁺]_i was evaluated from Fura-PE3 fluorescence emission measurements (510 nm) perpendicular to the excitation beam. The lower part of the transmitted light beam was blocked with an opaque panel in a specially designed cuvette holder so that the novel stirrer (which protrudes into the excitation beam) did not interfere with the aggregation measurements. The excitation wavelength alternated between 340 and 380 nm for the [Ca²⁺]_i measurements; transmitted light was monitored during the 380-nm excitation phase only. Aggregation is expressed as the change in light transmittance relative to the transmittance in the absence of platelets. This experimental approach yields aggregation data that are indistinguishable from data obtained with a traditional aggregometer.

Production of thromboxane A₂ was assessed in the samples used to monitor the platelet [Ca²⁺]_i signal. Platelets were incubated with 1 mg/mL ristocetin and 5 μg/mL human vWF for 10 minutes, a time sufficient for the [Ca²⁺]_i transient to be complete at 37°C. Incubations of the same duration were performed at the lower temperatures, and equivalent control incubations were conducted at each temperature with 1 mg/mL ristocetin in the absence of vWF. Analogous studies were conducted with 20 μmol/L arachidonic acid, except that the incubation time was 3 minutes. Each reaction was stopped by adding 1 mmol/L aspirin and 10 mmol/L EDTA (in buffered Tyrode’s solution); the supernatant fraction was collected after centrifugation for 1 minute at 16,000g. Thromboxane A₂ production during the incubation was assessed by competitive enzyme immunoassay of thromboxane B₂ (its stable breakdown product) in the supernatant fraction, using a commercial assay kit (Cayman Chemicals, Ann Arbor, MI). All thromboxane B₂ samples from studies on platelets from a particular donor were included in a single assay; the within-assay coefficient of variation for these assays averaged 4%.

Human platelets were fixed with 0.6% (wt/vol) paraformaldehyde by the method of Brinkhous and Read26 and washed extensively. Ristocetin-mediated agglutination of the fixed platelets by vWF was assessed by monitoring light transmittance at 380 nm (as for the aggregation measurements on live platelets).

Most data on platelet [Ca²⁺]_i and thromboxane A₂ production in this study were not normally distributed. Such data are thus presented as a median value with the interquartile range, and nonparametric tests were used to determine statistical significance (specific tests indicated in Results). Data on kd values for binding of calcium to Fura-PE3 were consistent with a normal distribution and are presented as a mean value ± standard error of mean (SEM).

The calcium-binding characteristics of Fura-PE3 were evaluated from excitation spectra measured in the presence of a series of free calcium concentrations. Three studies at 20°C yielded an average kd value of 251 ± 9 nmol/L (mean ± SEM) for Fura-PE3 at pH 7.0. A significantly lower kd value (146 ± 9 nmol/L,17 based on four studies; P < .001, Student’s t-test) was found at 37°C and pH 7.0.

Ristocetin-mediated binding of multimeric human vWF in the presence of extracellular calcium induced a gradual and transient platelet [Ca²⁺]_i increase at 37°C (Fig 2C). This [Ca²⁺]_i signal arose after an appreciable lag phase. At 20°C or 25°C, in contrast, vWF did not induce a perceptible increase in [Ca²⁺]_i (Fig 2A and Table 1). Studies at 30°C showed either a small increase in platelet [Ca²⁺]_i (Fig 2B) or no discernible increase (four of the eight studies). Analogous studies performed in the absence of extracellular calcium (with 1 mmol/L EGTA) showed the same pattern of temperature dependence (data not shown).

Stirring of the platelet suspension was required for vWF to induce a [Ca²⁺]_i increase at 37°C, suggesting that platelet aggregation might be necessary. The role of vWF binding to the glycoprotein IIb-IIIa complex (GP IIb-IIIa) in the signaling mechanism was evaluated using a monoclonal antibody against GP IIb-IIIa (10E5; kindly provided by Dr Barry Coller, Mount Sinai Medical Center, New York, NY).27 An antibody concentration of 10 μg/mL proved sufficient to prevent binding of vWF to GP IIb-IIIa; it abolished the aggregation by vWF of platelets that had been activated by U-46619 (Fig 3A). In contrast, preincubation with this concentration of antibody did not significantly affect the platelet [Ca²⁺]_i signal evoked by ristocetin-mediated binding of vWF at 37°C (Fig 3B and C). Median values (from four studies) for the peak [Ca²⁺]_i increase were 98 nmol/L in untreated platelets, 86 nmol/L in platelets pretreated with an isotype-matched control Ig (IgG2a), and 89 nmol/L after treatment with 10E5 antibody (P > .2, Friedman’s test). Blockade of GP IIb-IIIa with the tetrapeptide Arg-Gly-Asp-Ser (1 mmol/L) also had negligible effect on the vWF-induced [Ca²⁺]_i signal.17These data imply that binding of vWF to GP Ib-IX-V triggers the platelet [Ca²⁺]_i increase.

The absence of a vWF-induced platelet [Ca²⁺]_isignal at the lower temperatures could reflect a strong temperature dependence for either binding of vWF to the GP Ib-IX-V complex or a subsequent step in the signal transduction process triggered by vWF. These two possibilities were discriminated primarily by evaluating the temperature dependence of ristocetin-mediated platelet aggregation by vWF. (“Aggregation” is used here in an inclusive sense to encompass both physical agglutination through binding of vWF to different platelets and true aggregation involving platelet activation. Both processes occur when live platelets are incubated with vWF in the presence of ristocetin, and the measurement of light transmittance cannot distinguish between them.) Aggregation was assessed simultaneously with the platelet [Ca²⁺]_imeasurements by using a novel configuration of the spectrofluorometer (Fig 1). The aggregation of live platelets by vWF was essentially unaffected by temperature over the range 20°C to 37°C (Fig 4A and B). Temperature also had negligible effect when aggregation was assessed in the presence of Arg-Gly-Asp-Ser (data not shown). Ristocetin-mediated agglutination of fixed platelets by vWF declined marginally when the temperature was lowered from 37°C to 20°C (Fig 4C and D), but there was no significant reduction at 30°C (data not shown). These data together imply that the marked temperature dependence of the vWF-induced platelet [Ca²⁺]_i signal cannot be attributed to differences in vWF binding to GP Ib-IX-V.

The influence of temperature on the [Ca²⁺]_isignal was also evaluated for other platelet agonists. For the stable thromboxane A₂ analog U-46619, the amplitude of the agonist-induced [Ca²⁺]_i transient was not significantly affected by temperature (Fig5 and Table 1; P > .2, Friedman’s test). For α-thrombin and ADP, the amplitude of the [Ca²⁺]_i signal tended to diminish as the temperature was increased from 20°C to 37°C (Table 1); this diminution was marked for α-thrombin, but modest for ADP. The time course of the [Ca²⁺]_i transient for each of these three agonists tended to be prolonged at the lower temperatures (Fig 5). Studies with different concentrations of U-46619 (0.05 to 1 μmol/L) and α-thrombin (0.01 to 0.1 U/mL) yielded similar observations (data not shown). Resting platelet [Ca²⁺]_i rose gradually with increasing temperature (Table 1). Analogous findings were made in studies conducted with these agonists in the absence of extracellular calcium (data not shown).

The platelet [Ca²⁺]_i signal induced by arachidonic acid showed a markedly different pattern of temperature dependence than these direct platelet agonists. A robust [Ca²⁺]_i increase was observed at 37°C in the presence of extracellular calcium (Fig6C), a very modest increase generally occurred at 30°C (Fig 6B), and no [Ca²⁺]_i signal was seen at 20°C and 25°C (Fig 6A). The same pattern was observed in the absence of extracellular calcium (data not shown). This pattern of temperature dependence was closely analogous to that observed with vWF (Table1).

The strong temperature dependence of the platelet [Ca²⁺]_i signal induced by arachidonic acid, but lack of such dependence when U-46619 was the agonist, suggested that metabolism of arachidonic acid by the cyclo-oxygenase pathway might be severely curtailed at the lower temperatures. This possibility was assessed directly by assay of thromboxane A₂ production in platelets incubated with exogenous arachidonic acid at different temperatures. Metabolism of arachidonic acid to thromboxane A₂ was substantially abrogated when the incubation temperature was reduced from 37°C to 30°C (Fig 7A; P < .01, Friedman’s test with a nonparametric Student-Newman-Keuls posthoc test) and was virtually abolished at the lower temperatures (20°C and 25°C).

The similar patterns of temperature dependence for platelet [Ca²⁺]_i signaling with vWF and arachidonic acid suggested that these signals might arise through similar mechanisms. Assay of thromboxane A₂ production in response to the interaction of vWF with the platelet provided an independent test of this hypothesis. Ristocetin-mediated binding of vWF promoted a substantial increase in platelet thromboxane A₂ production at 37°C (Fig 7B). vWF-induced thromboxane A₂ production was substantially diminished at 30°C (P < .01) and abolished at 20°C and 25°C (Fig 7B). This pattern of temperature dependence closely matches the patterns seen for thromboxane A₂ production with arachidonic acid and for platelet [Ca²⁺]_i signaling with both vWF and arachidonic acid.

If thromboxane A₂ plays an essential role in the mechanism by which vWF elevates platelet [Ca²⁺]_i, treatment with a cyclo-oxygenase inhibitor should abolish the [Ca²⁺]_i signal. Studies were conducted in aspirin-treated platelets to evaluate this possibility. Pretreatment with 0.2 mmol/L aspirin consistently abrogated the platelet [Ca²⁺]_i increase induced by ristocetin-mediated binding of vWF at 37°C (Fig 8A and B; P < .005, Wilcoxon signed-rank test). Such treatment also abolished vWF-induced thromboxane A₂ production (Fig 8C; P < .005). Platelet [Ca²⁺]_i signaling and thromboxane A₂ production in response to 20 μmol/L arachidonic acid were also eliminated (data not shown), confirming the effectiveness of cyclo-oxygenase inhibition.

Temperature has only a modest influence on the amplitude and kinetics of [Ca²⁺]_i signals in response to most agonists in many types of cell.20,28,29 The limited nature of this influence has served as a rationale for performing some [Ca²⁺]_i signaling studies at ambient temperature when the demands of a particular experimental approach make it technically difficult to perform the study at a physiological temperature. For this reason, confocal microscopic evaluation of [Ca²⁺]_i signals in individual cells is often performed at ambient temperature.30-32 This rationale also explains the choice of an ambient temperature for studies of platelet [Ca²⁺]_i signaling under shear stress.10,12,13 

The limited influence of temperature on [Ca²⁺]_i signals induced by the classical platelet agonists U-46619, α-thrombin, and ADP in the present study (Fig 5 and Table 1) is consistent with the patterns reported previously for α-thrombin and ADP in the platelet33,34 and for many agonists in various cells.20,28,29 The platelet [Ca²⁺]_i transients with these classical agonists tended to be somewhat prolonged at the lower temperatures. This pattern parallels previous reports of a slower removal of excess calcium from the cytosol at lower temperatures.35,36U-46619 triggers a platelet [Ca²⁺]_i increase primarily through stimulation of phospholipase C and inositol 1,4,5-trisphosphate-mediated calcium release from the intracellular stores,37 whereas influx of calcium through receptor-operated channels in the plasma membrane is thought to play the main role in ADP-induced [Ca²⁺]_isignaling.38,39 The amplitude of an agonist-induced [Ca²⁺]_i transient reflects a balance between calcium entry into the cytosol and its extrusion. That the [Ca²⁺]_i signal with U-46619 is temperature invariant might be related to the enzyme-mediated nature of both calcium release from the intracellular stores and its extrusion from the cytosol. A lesser effect of temperature on calcium influx with ADP (a diffusion-limited process) than on extrusion might also explain the modest increase in amplitude of its [Ca²⁺]_isignal at lower temperatures. The pattern of inverse temperature dependence with α-thrombin, however, cannot be rationalized so readily.

In striking contrast to our findings with U-46619, ADP, and α-thrombin, the platelet [Ca²⁺]_i signal induced by ristocetin-mediated binding of vWF was inhibited substantially by a reduction in temperature to 30°C and abrogated completely by a further reduction to 20°C or 25°C (Fig 2 and Table 1). This marked difference in temperature dependence strongly suggests a substantive difference in the signaling mechanism for vWF compared with the other agonists. The protracted time course of the [Ca²⁺]_i signal with vWF (at 37°C) also supports such a difference. In particular, the pronounced lag phase between addition of vWF and the platelet [Ca²⁺]_i increase argues in favor of an indirect signaling mechanism for vWF. Furthermore, the similarity in temperature dependence of the platelet [Ca²⁺]_i signals with vWF (Fig 2) and arachidonic acid (Fig 6) suggests that release of arachidonic acid may be a proximal event in [Ca²⁺]_i signaling with vWF. The finding that vWF promotes thromboxane A₂production in the platelet (Fig 7B) lends further support to this possibility. Moreover, the abolition of the [Ca²⁺]_i signal by the cyclo-oxygenase inhibitor aspirin (Fig 8A and B) provides direct evidence that thromboxane A₂ plays an essential role in the signaling mechanism for vWF. Metabolism of arachidonic acid to thromboxane A₂ by the cyclo-oxygenase pathway may exhibit such a strong temperature dependence (Fig 7A) because it involves three catalytic steps, cyclization and peroxidation by cyclo-oxygenase (prostaglandin H synthase) and isomerization by thromboxane A synthase. The temperature dependence of these steps, in combination, can explain the marked temperature dependence for platelet [Ca²⁺]_isignaling with vWF.

Prior studies of vWF-induced [Ca²⁺]_isignaling in the platelet have yielded inconsistent findings. One of the earliest studies by Kroll et al,11 using ristocetin to trigger vWF binding, led its authors to postulate that activation of the phospholipase A₂ pathway and subsequent production of thromboxane A₂ play a major role in vWF-induced [Ca²⁺]_i signaling. There have been no further detailed studies of the signaling mechanism using this experimental approach. However, one subsequent study, using a mutant type 2B vWF that binds spontaneously to platelet GP Ib-IX-V, yielded data that broadly support this mechanism.19 In contrast, other investigators have suggested that the proximal event in vWF signaling leading to a [Ca²⁺]_i increase is an influx of calcium through channels in the plasma membrane1,10,12,13,16,40; these investigators contend that thromboxane A₂ does not contribute significantly to the process. Support for the latter mechanism is derived largely from dynamic studies where shear stress acted as the trigger for vWF binding to platelet GP Ib-IX-V.10,12,13 One study with a static system, measuring the response to spontaneous binding of porcine vWF, has also lent support to this mechanism.16 The putative ability of vWF to signal by two distinct mechanisms when its interaction with platelet GP Ib-IX-V is instigated by different means has not yet been explained. It does, nonetheless, present an attractive therapeutic target for prevention of pathological thrombosis without impairment of normal hemostasis.

It is not uncommon for a signal transduction pathway to bifurcate downstream of a receptor.41,42 Such bifurcation could enable platelet GP Ib-IX-V, when occupied by vWF, to signal through both phospholipase A₂ and a plasma membrane calcium channel. This would provide a partial explanation for the putative ability of vWF to stimulate a different primary signaling pathway when its interaction with GP Ib-IX-V is initiated by different means. This hypothesis, however, does not explain why one signaling pathway should predominate (in general) under static conditions, but a different pathway under shear stress.

There is not yet sufficient evidence to conclude that the signaling mechanism for vWF really differs in this manner. Instead, a more mundane explanation for the divergent findings among prior studies should, perhaps, be sought. Could the discrepancies simply reflect technical differences between the various experimental approaches? The present study does provide a simple technical explanation for the apparent lack of involvement of the phospholipase A₂pathway in signaling for vWF under shear stress. Prior investigations into platelet [Ca²⁺]_i signaling under shear stress appear to have been conducted at room temperature.10,12,13 Our study shows that arachidonic acid effectively cannot be metabolized to thromboxane A₂ (a necessary step in the phospholipase A₂ pathway) in the platelet at this temperature (Figs 6 and 7). Although the isolated cyclo-oxygenase enzyme has demonstrable activity at 25°C,43 this need not pertain in the cellular milieu where other competing reactions and regulatory mechanisms exist.44 Clearly, further studies of vWF-induced [Ca²⁺]_i signaling under shear stress must be undertaken at 37°C to establish unequivocally whether the phospholipase A₂ pathway plays a significant role under these circumstances. It is worth noting, in this context, that the classical studies of vWF-mediated platelet adhesion and aggregation on exposed vascular subendothelium in a Baumgartner chamber showed a strong temperature dependence for platelet activation under shear stress.45 Our earlier studies, moreover, have raised doubts about the validity of most [Ca²⁺]_imeasurements in aggregating platelets.17,46,47 As these doubts apply particularly to studies where a [Ca²⁺]_i signal is observed only in the presence of extracellular calcium (Kermode et al, manuscript submitted), the apparent ability of vWF to induce calcium influx into the platelet (under any circumstances) should be questioned. It is possible that the prior studies of vWF-induced [Ca²⁺]_i signaling under shear stress failed to uncover the real signaling pathway because they were performed at ambient temperature, but, instead, reported an artifactual [Ca²⁺]_i increase due to platelet aggregation.

In conclusion, platelet [Ca²⁺]_i signaling induced by ristocetin-mediated interaction of vWF with GP Ib-IX-V is markedly influenced by temperature. A similar pattern of temperature dependence is seen for [Ca²⁺]_i signaling with arachidonic acid, but not with other platelet agonists. Treatment of platelets with the cyclo-oxygenase inhibitor aspirin abolishes the vWF-induced [Ca²⁺]_i signal. These findings are consistent with the concept that stimulation of phospholipase A₂ is a necessary and proximal event in platelet [Ca²⁺]_i signaling with vWF.

We are greatly indebted to Joe Ed Smith for his expertise in constructing the cuvette holder and novel stirrer (Fig 1). The generous gift of the 10E5 antibody by Dr Barry Coller is most appreciated. Thanks are due to Dr Rodney Baker for his critical review of the manuscript. We are also grateful to the volunteer donors who provided blood samples for these studies.