Interaction of von Willebrand factor (vWF) with the platelet is essential to hemostasis when vascular injury occurs. This interaction elevates the intracellular free calcium concentration ([Ca2+]i) and promotes platelet activation. The present study investigated the temperature dependence of vWF-induced [Ca2+]i signaling in human platelets. The influence of temperature can provide invaluable insight into the underlying mechanism. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of vWF induced a transient platelet [Ca2+]i increase at 37°C, but no response at lower temperatures (20°C to 25°C). This temperature dependence could not be attributed to a reduction in vWF binding, as ristocetin-mediated platelet aggregation and agglutination were essentially unaffected by temperature. Most other platelet agonists (U-46619, -thrombin, and adenosine 5′-diphosphate [ADP]) induced a [Ca2+]isignal whose amplitude did not diminish at lower temperatures. The [Ca2+]i signal in response to arachidonic acid, however, showed similar temperature dependence to that seen with vWF. Assessment of thromboxane A2 production showed a strong temperature dependence for metabolism of arachidonic acid by the cyclo-oxygenase pathway. vWF induced thromboxane A2production in the platelet. Aspirin treatment abolished the vWF-induced [Ca2+]i signal. These observations suggest that release of arachidonic acid and its conversion to thromboxane A2 play a central role in vWF-mediated [Ca2+]i signaling in the platelet at physiological temperatures.

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Letters to the Editor difficult to compare these two observations because the IL Test (Instrumentation Laboratory) used by Sorice et al. to measure PS activity presents many differences when compared with the Stago assay we have used. The difference of reagents could lead to a variable sensitivity of both tests to inhibition of PS activity by PS autoantibodies. It is known that the presence of anti-B2GPI autoantibodies with ACA and LA in SLE is a biological state specially liable to thrombotic complications (6). The suggestion was made that acquired free PS deficiency could represent a major mechanism for the development of thrombosis in SLE patients with antiphospholipid antibodies (aPLs) (7). In our case it does not play a decisive role as it appeared only after the thrombotic event. However, the involvement of anti-PS autoantibodies in the thrombosis could not be ruled off, indeed it could act locally at a low level without affecting the free PS level. The co-occurrence of aPL, antiPS and anti-B2GPI autoantibodies in this clinical context is probably not fortuitous: according to Roubey and others, the case herein could be related to the syndrome due to autoantibodies to phospholipid binding proteins (8). These autoantibodies could belong to the same family with varying affinities for protein-phospholipid complexes. By interfering with the function of proteins involved in the coagulation pathway, they could explain tluombotic tendencies in some patients. In our case, heterozygocity for FV: Q506 could have potentiahzed the thrombotic risk. A further large study is needed to evaluate the prevalence of anti-PS autoantibodies in SLE and their predictive value for a thrombotic event in comparison with aPLs and anti-$2GPI autoantibodies.

SummaryInteraction of von Willebrand factor (VWF) with the platelet promotes hemostasis upon vascular injury. Such interaction raises intracellular free calcium concentration ([Ca2+]i) and induces platelet activation. The platelet [Ca2+]i increase is generally attributed to influx across the plasma membrane. The present study defined the contribution of intracellular calcium stores. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of VWF transiently elevated [Ca2+]i after a lag phase. Studies with 63 healthy donors consistently revealed a VWF-induced platelet [Ca2+]i signal in the absence of extracellular calcium; there was only modest enhancement with extracellular calcium. Blockade of plasma membrane calcium channels did not diminish the signal, whereas depletion or blockade of the intracellular calcium stores abolished it. These findings imply that release from intracellular stores is responsible for the VWF-induced platelet [Ca2+]i increase. Influx across the plasma membrane plays no more than a minor role, probably representing “capacitative entry” to refill the intracellular stores.