Cell-Cell Interactions in Thrombosis: Modulation of Platelet Function and Possibilities of Pharmacological Control with Aspirin Ma Teresa SANTOS, Juana VALLÉS, Justo AZNAR Department of Clinical Pathology and Research Center, La Fe University Hospital, Valencia, SPAIN Turk J Haematol 2002;19(2):103-111 Thrombosis is a complex and dynamic phenome- bus growth[3-5]. To study these processes, we have de- non, involving interaction of endothelial and blood veloped an experimental procedure that allows activa- cells, plasma clotting, fibrinolysis and hemorrheologic tion and platelet recruitment to be assessed separa- factors. Platelets play an essential role in the process of tely[4-6]. thrombosis[1]. Interaction of platelets with collagen and/or thrombin initiates a complex biochemical sequ- Platelets, leukocytes and erythrocytes appear in the [7] ence of signal transmission resulting in a functional thrombus . This might indicate that the interaction of cell response. platelets with other blood cells is important in regulati- on of the process of thrombosis. Platelet–leukocyte in- There are several signal mechanisms involved in teraction inhibits platelet reactivity, whereas erythrocy- platelet activation, including specific receptors, G pro- te–platelet interaction enhances it[3-6]. It has also been teins, phosphatidyl inositol metabolism, synthesis of shown that this cell interaction alters the antithrom- thromboxane A2 (TXA2) and other eicosanoids, chan- bocytic effect of aspirin and dipyridamole[4-15]. ges in cytosolic calcium, and protein phosphorylation, in serine/threonine and tyrosine residues[2]. These sig- A well-documented example of this intercellular nal mechanisms result in a conformational change in interaction is the transcellular metabolism of eicosano- the GP IIb IIIa receptor, platelet secretion, platelet–pla- ids, which occurs among different blood cells, and bet- telet aggregation, exposure to procoagulant phospholi- ween blood and endothelial cells[16,17]. In this process, pids in the membrane, clot retraction, and synthesis of arachidonic acid, compounds of their intermediate me- different metabolic products. tabolism or the specific final products of the metabo- lism of one cell are changed by another one into speci- We have found that the products released by acti- fic products of the second, or into new compounds vated platelets have a proaggregating action on the tar- which neither cell can synthesize in an independent get platelets, favoring platelet recruitment and throm- manner[16]. Metabolic cooperation in the synthesis of 103 Cell-Cell Interactions in Thrombosis: Modulation of Platelet Function and Santos MT, Vallés J, Aznar J. Possibilities of Pharmacological Control with Aspirin lipid mediators occurs through various cellular interac- mediated by platelet P-selectin leading to the expressi- tions, including interactivus between platelets and en- on of tissue factor in leukocytes[30,44,48]. dothelial cells, platelets and neutrophils, platelets and Role of erythrocytes in hemostasis and thrombosis: erythrocytes, endothelial cells and neutrophils, and ne- Erythrocytes (RBCs) also play a role in thrombogene- utrophils and erythrocytes[18-23]. sis[10,49]. This was first reported by Duke in 1910, who Platelet–neutrophil interaction: Experimental stu- found that the prolonged bleeding times in anemic pa- dies suggest that polymorphonuclear neutrophils tients became normal after the hematocrit was incre- (PMNs) play a role in thrombosis[24]. It is thought that ased[50]. This clinical association between bleeding ti- PMNs play a role in vascular disease through their ca- mes and the hematocrit were confirmed by other aut- pacity to adhere to endothelial cells and aggregate du- hors[51-54]. Erythrocytes have also been involved in at- ring acute inflammatory processes[25,26]. Activated herothrombotic processes and associated with establis- PMNs release into the micro environment azurophillic hed risk factors such as diabetes, hypercholesterole- granules, rich in proteases, and oxygen radicals, which mia, and hypertension[51,55-63]. may damage endothelial cells, disrupting their throm- Erythrocytes may participate in thrombus formati- boregulating function[27-29]. Moreover, PMNs partici- on, changing the hemorrheologic properties of blood, pate in thrombotic events by contributing to coagulant modulating the antiaggregating action of the blood ves- processes and by modulating different stages of the sels, inactivating prostacyclin, eliminating NO (EDRF) process of thrombosis[30]. and influencing thrombocytic function, although it has There are conflicting reports on the role of PMNs no effect on the ecto-ADP loops of the vascular endot- in the process of thrombosis[31]. Some authors report helium[64-72]. that PMNs stimulate platelet activation, whereas others Effect of the erythrocytes on thrombocytic reacti- report inhibition by PMNs[6,12,32-38]. These conflicting vity: Erythrocytes act on platelets by physical and bi- results may arise from differences in experimental con- ochemical mechanisms[73,74]. Hellem and Gaarder re- ditions between studies, in particular whether PMNs ported for the first time in 1961 on the capacity of are studied at rest or after stimulation and whether erythrocytes to increase thrombocytic function; in this whole cells or isolated compounds derived from stimu- study, platelet adhesiveness to glass increased in the lated leukocytes, such as the cathepsin G or oxidant presence of erythrocytes[75]. These authors suggested products, are assessed. Various mechanisms by which that ADP released by lysis or cellular microlysis of PMNs might inhibit platelet function have been sug- erythrocytes could be the biochemical factor respon- gested: Synthesizing nitric oxide (NO), modulating sible for increased adhesion-aggregation of platelets in synthesis of eicosanoids, modifying glycoproteins of the presence of erythrocytes[76]. It was later shown that the platelet membrane, generating H O and other oxi- 2 2 erythrocytes increase platelet adhesion and thrombus dants, membrane ADP-arg, or other mecha- formation in damaged endothelium[77]. They correct nisms[6,12,34-42]. Physical proximity between cells is adhesion defects in patients with “storage pool defici- favored by P-selectin, an adhesion protein exposed on ency (SPD)” depending on the hematocrit, they incre- the membrane of activated platelets, which binds to a ase spontaneous platelet aggregation, enhancing plate- specific receptor in the PMNs, PSGP-1, and between let aggregation due to collagen, they increase platelet the GP IIb IIIa on platelets and ß integrin on 2 activation and recruitment and they shorten the forma- PMNs[33,43,44]. tion time of the thrombus in the platelet function analy- Aspirin amplifies the inhibiting effect of PMNs on zer PFA-100[4,5,14,63,78-82]. thrombocytic reactivity, although it does not change The biochemical mechanisms that regulate the ef- the capacity of the platelets to expose P-selec- fects of erythrocytes on platelet function are not well tin[6,12,13,37,45-47]. Thus, aspirin may not modify other known. We have shown that erythrocyte–platelet inte- processes depending on P-selectin that are important in raction increases in the presence of cellular stimulati- the PMN-platelet interaction, such as the recruitment on, both by ADP concentration (7 times) and ATP (5 ti- of PMNs by activated platelets to the forming throm- mes); this occurs without erythrocyte lysis[5]. It is bus in the area of vascular damage, or fibrin formation 104 Turk J Haematol 2002;19(2):103-111 Cell-Cell Interactions in Thrombosis: Modulation of Platelet Function and Possibilities of Pharmacological Control with Aspirin Santos MT, Vallés J, Aznar J. worth mentioning that the potentiating effect of eryth- aspirin is not protective in a high proportion of pati- rocytes on the erythrocyte reactivity requires the simul- ents, however, and new therapies are needed to reduce taneous presence of activated platelets and intact eryth- thrombotic risk in these patients. rocytes[4,5]. The prothrombotic effect of erythrocytes Aspirin inhibits synthesis of TXA in an irreversib- does not occur in the presence of platelet stimulation or 2 le manner[96]. Thrombotic events in patients treated erythrocyte lysis. This suggests that, under experimen- with aspirin could be due to biochemical mechanisms tal conditions, the effect of erythrocytes is related mo- of platelets that surpass the aspirin effect. This might re to metabolic than physical factors, requiring the cel- occur because of exposure of platelets to high concent- lular integrity of the erythrocyte. rations of collagen or thrombin, or possibly ADP. Un- Erythrocyte–platelet interaction increases hydroly- der these conditions, platelets do not require the amp- sis of the arachidonic acid of the phospholipids, as well lification action of thromboxane to produce a proagg- as the metabolism of the platelet enzymes cyclooxyge- regation response, i.e., platelet activation is stimulated nase and lipooxygenase[4]. This interaction increases by cyclooxygenase 1 (COX-1) independent mecha- the release of eicosanoids and arachidonic acid to the nisms[97,98]. cellular microenvironment, which could stimulate ot- We have found experimental evidence that aspirin her platelets and provide the substratum needed for the not only acts on platelets by inhibiting COX-1, but al- transcellular metabolism with other blood cells. An so exerts a direct effect on erythrocytes, reducing their example of this