SummaryThe adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counterreceptor (CD 15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein III a which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

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Acetylsalicylic acid is widely used in the primary and secondary prevention of cardiovascular diseases. In the current study, we used platelet aggregation ex vivo in platelet-rich plasma induced with arachidonic acid as a routine method for the determination of the individual dose of acetylsalicylic acid necessary to inhibit platelet aggregation in 108 patients with cardiovascular diseases. In 40% of all patients studied, a dose of 30 mg/day was sufficient to block the arachidonic acid-induced platelet aggregation nearly completely. In 50% of all patients, a dose of 100 mg/day was necessary. In 10% of all patients, the dose had to be further increased to 300 mg/day or even to 500 mg/day to inhibit platelet aggregation nearly completely. These results demonstrate that platelet aggregation can be used as a simple routine laboratory method to control acetylsalicylic acid treatment in patients with cardiovascular diseases and to determine individual doses of acetylsalicylic acid for a nearly complete inhibition of platelet aggregation. With a standard dose of 100 mg/day, 10% of the patients were nonresponders.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.

Adhesion of platelets to neutrophils and monocytes is believed to play an important role in intercellular communication. Evidence has been provided that such heterotypic cell-cell contacts via adhesion molecules may be directly involved in intercellular signal transduction as well as facilitate the action of soluble signal transmitters, e.g. cathepsin G, PAF or nitric oxide. With respect to platelet activation, stimulatory and inhibitory effects of leukocytes have been reported, and the results obtained seem to be influenced by the experimental conditions. We investigated the effect of leukocyte stimulation on platelet behaviour in samples of human citrated whole blood. Adding the chemotactic peptide FM LP, which stimulates neutrophils and monocytes but not lymphocytes and platelets, to stirred whole blood samples resulted in a significant enhancement ( P < 0.01) of spontaneous as well as ADP-induced platelet aggregation (25 vs 33% and 66 vs 69% , respectively). In contrast stirring-induced as well as ADP-induced increase of P-selectin exposure (33 and 107% , respectively) was not affected by FMLP. In unstirred whole blood samples, about 10 to 20% of neutrophils and monocytes had bound platelets to their surfaces, and the number of these heterotypic conjugates was enhanced about twofold during spontaneous platelet aggregation. Addition of FMLP significantly reduced the stirring-induced formation of platelet-neutrophil conjugates but not of platelet-monocyte conjugates. These results indicate that neutrophil and/or monocyte activation in whole blood may enhance platelet aggregation, but not secretion (CD62P exposure) and formation of heterotypic platelet-leukocyte conjugates.