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This review article describes the different receptors, second-messengers and mechanisms involved in platelet activation. Several platelet agonists have well-defined receptors at the platelet membrane of which a number are single polypeptides with 7 hydrophobic transmembrane domains. These receptors are connected, via GTP regulatory proteins, with cytoplasmic second-messenger-generating enzymes. Phospholipase C and adenylate cyclase are the two best-known enzymes, generating inositol triphosphate (IP3) and diacyl glycerol from phosphatidylinositol biphosphate and cyclic AMP from ATP respectively. The intraplatelet free calcium level, which is critical for the activation status of the platelet, is increased by IP3 and is lowered in the presence of rising cyclic AMP concentrations. Shape-change occurs with small increases in intraplatelet calcium, while aggregation and secretion of granules take place at higher calcium, levels. The role of myosin and actin filaments and of transmembrane glycoproteins is further discussed.

SummaryIn May 1990, 218 patients with haemophilia A regularly attending the Leuven Haemophilia Center were randomly assigned to a group receiving either of two newly introduced factor VIII concentrates: factor VIII-P, an intermediate purity pasteurized concentrate, or factor VIII-SD, a high purity concentrate treated with solvent-detergent for viral inactivation.Patients were followed from May 1990 until October 1991. Between August 1991 and October 1991 a clinically important factor VIII inhibitor was detected in five out of the 109 patients receiving factor VIII-P while none of the 109 patients receiving factor VIII-SD developed such antibodies. All patients acquiring an inhibitor had previously been clinically tolerant to transfused factor VIII with 200 to more than 1,000 days of exposure to factor VIII prior to May 1990. Patients with inhibitors were transfused daily with 30 U factor VIII-SD per kg body weight, which was associated with a gradual decline of the inhibitor level. In all patients the antibodies were relatively slow-acting and predominantly directed towards the light chain of factor VIII.This study demonstrates a higher than expected incidence of factor VIII inhibitors associated with the use of a specific factor VIII concentrate in multitransfused haemophilia A patients. It indicates the usefulness of evaluating newly introduced concentrates in prospective, randomized trials.

Short-term increases in particulate air pollution are associated with increased incidence of cardiovascular events. Previously, we showed that intratracheally instilled diesel exhaust particles (DEPs) are prothrombotic. Here, we investigated the time course and the mechanisms. At 1, 6, and 24 hours after instillation of 50 microg DEPs per hamster, the mean size of in vivo-induced and quantified venous thrombosis was increased by 480%, 770%, and 460%, respectively. Platelets activation in blood was confirmed by a shortened closure time in the platelet function analyzer (PFA-100). In bronchoalveolar lavage, neutrophils and histamine levels were increased at all time points. In plasma, histamine was increased at 6 and 24 hours but not at 1 and 3 hours. Pretreatment with a histamine H1-receptor antagonist (diphenhydramine, 30 mg/kg intraperitoneally) abolished the DEP-induced neutrophil influx in bronchoalveolar lavage at all time points. However, diphenhydramine pretreatment did not affect DEP-induced thrombosis or platelet activation at 1 hour, whereas both were markedly reduced at 6 and 24 hours. In conclusion, pulmonary inflammation and peripheral thrombosis are correlated at 6 and 24 hours, but at 1 hour, the prothrombotic effects do not appear to result from pulmonary inflammation but possibly from the blood penetration of DEP-associated components or by DEP particles themselves.

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1 ؊/؊ mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1 ؊/؊ platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1 ؊/؊ vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1 ؊/؊ vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1 ؊/؊ mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1 ؊/؊ venules (1027 ؎ 377 seconds) and arterioles (858 ؎ 289 seconds) than in WT vessels (559 ؎ 241 seconds, P < .001; 443 ؎ 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local plateletreleased TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively. (Blood. 2006;107: 955-964)

We have generated transgenic mice overexpressing the human P2X 1 ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X 1 ionotropic activity as determined by more prominent P2X 1 -mediated Ca 2؉ influx and platelet shape change. P2X 1 overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A 2 mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds ؊1 resulted in increased P2X 1 -dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X 1 ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A 2 -, and shear stress-triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes. (Blood. 2003;

We previously showed that ␤ 2 -glycoprotein I (␤ 2 GPI)-dependent lupus anticoagulants (LAs) form bivalent antigen-antibody complexes with high affinity for phospholipids; these complexes are responsible for their in vitro anticoagulant effect. We now studied the role of these bivalent complexes in arterial thrombosis in the hamster. Three monoclonal antibodies (mAbs) raised against human ␤ 2 GPI were selected on the basis of their crossreactivity with hamster ␤ 2 GPI. Two of these, one with LA activity, 5H2, and one with only anticardiolipin properties, 11E8, were infused at 0 to 10 mg/kg prior to photochemically induced vessel damage. 5H2 promoted thrombus formation dose dependently, raising the thrombus size from 6.0 arbitrary units (AU) in controls (n ‫؍‬ 9) to 65.0 AU in the high-dose group (10 mg/kg, n ‫؍‬ 6, P ‫؍‬ .007). The LA ؊ mAb 11E8 and mAb 27A8, reactive with human ␤ 2 GPI exclusively, did not significantly promote thrombus formation. In a second set of experiments, intact mAb 5H2 was compared to its fragments. Intact mAb 5H2 at 3.3 mg/kg and the equimolar dose of F(ab) 2 fragments (2.2 mg/kg) promoted thrombus formation equally well (55.8 AU, n ‫؍‬ 8 and 62.5 AU, n ‫؍‬ 7, respectively); mAb 5H2-derived Fab fragments were inactive. Immunohistochemical analysis showed platelet-rich thrombi, with 5H2 or its F(ab) 2 fragments mainly bound to individual platelets. Our results indicate that bivalent immune complex formation plays an important role in the genesis of arterial thrombosis by certain antiphospholipid antibodies. Cellular activation via the Fc portion of these immune complexes, however, is not essential, because F(ab) 2 fragments of 5H2 still promote thrombus formation. (Blood. 2003;

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3-month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n ‫؍‬ 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n ‫؍‬ 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n ‫؍‬ 7 and 5, respectively). In vitro, endothelial VWF-platelet glycoprotein (GP) Ib and platelet P-selectinendothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n ‫؍‬ 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIb␣, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIb␣, and P-selectin to lesion-prone sites, before lesions are detectable. (Blood. 2002; 99:4486-4493)