Platelet activation by oxidized low density lipoprotein is mediated by CD36 and scavenger receptor-A.

OBJECTIVE: The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2' (apoER2')-mediated signaling to p38(MAPK) and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL. METHODS AND RESULTS: Oxidation of nLDL increases p38(MAPK) activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38(MAPK) activation by oxLDL but combined blockade inhibits p38(MAPK) by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38(MAPK) activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38(MAPK) by >70%. CONCLUSION: These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

Impaired platelet adhesion to lysed fibrin, whereas neutrophil adhesion remains intact under conditions of flow.

Vessel wall injury induces the formation of a haemostatic plug. Restoration of vascular integrity should involve cessation of further platelet and fibrin deposition and subsequent removal of these thrombi by both the fibrinolytic system and proteases delivered by infiltrating inflammatory cells. We hypothesized that adhesion of platelets and inflammatory cells [polymorphonuclear leucocyte (PMN)] to fibrin is differently supported after exposure of fibrin during fibrinolysis. Fibrin surfaces were exposed to fibrinolytic agents, and platelet and PMN adhesion was studied under conditions of flow. Specific adhesion of platelets to preformed fibrin was reduced by fibrinolytic treatment of the fibrin. PMN adhesion to fibrin was only slightly affected even after 180 min exposure to plasmin. With fibrin still present after fibrinolytic treatment, the impaired platelet adhesion seems explained by loss of the primary platelet adhesion site gamma400-411 on fibrin. PMN binding to fibrin clearly depends on other sites that are less degraded by fibrinolysis. We have shown that PMN adhesion in flowing blood to lysed fibrin was still present, whereas platelet adhesion was impaired due to the loss of the primary platelet adhesion site gamma400-411. Based on our in-vitro perfusion model, we conclude that fibrinolysis specifically interferes with the thrombogenicity of fibrin in the haemostatic plug, whereas the inflammatory response is preserved. The latter may participate in the long-term removal and restructuration of the plug.

Role of ADP receptor P2Y(12) in platelet adhesion and thrombus formation in flowing blood.

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y(1) and P2Y(12). We have investigated the role of P2Y(12) in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y(12). Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient's thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y(12) antagonist, N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y(1) antagonist (adenosine-3',5'-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y(12) interaction is essential for normal thrombus buildup on collagen. The patient's blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3',5'-diphospate. This suggested that P2Y(12) and P2Y(1) were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.

The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.

Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis.

BACKGROUND: The platelet adenosine 5'-diphosphate (ADP) receptor P2Y(12) plays a crucial role in haemostasis. Only a few patients with haemorrhagic diathesis due to molecular defects in the P2Y(12) receptor have been described so far. We report a novel molecular defect in the gene coding for P2Y(12) in a patient with a history of epistaxis, easy bruising and excessive posttraumatic blood loss. METHODS: Platelet aggregation studies, perfusion studies, in which patient blood was perfused over collagen surfaces at arterial shear rates, and PCR and sequencing were used. RESULTS: Platelet aggregation studies showed impaired ADP and collagen-induced aggregation for patient G.S. Perfusion of patient blood over collagen surfaces showed small thrombi consisting of spread platelets overlayered with non-spread platelets. These thrombi were identical to control thrombi formed in the presence of a P2Y(12) antagonist. DNA analysis of the P2Y(12) gene revealed a novel heterozygous base pair C-->A substitution in exon 3, changing codon 258 from proline to threonine in the third extracellular loop of the P2Y(12) receptor. CONCLUSIONS: We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment gamma316-322, part of the fibrin-specific region.

The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the gamma-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the gamma-chain segments gamma318-320 and gamma408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to gammaDelta408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: gammaDelta319-320, gamma318Asp-->Ala and gamma320Asp-->Ala. Furthermore, antibodies raised against the sequences gamma308-322 and gamma316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment gamma316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression.

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.

Thrombopoietin increases platelet adhesion under flow and decreases rolling.

Thrombopoietin (TPO) is known to sensitize platelets to other agonists at 20 ng/ml, and above 100 ng/ml it is an independent activator of aggregation and secretion. In studies with a perfusion chamber, TPO, between 0.01 ng/ml and 1 ng/ml, increased platelet adhesion to surface-coated fibrinogen, fibronectin and von Willebrand Factor (VWF) but not to a collagen-coated surface. Increased adhesion was observed at shear rates of 300/s and 800/s in perfusions with whole blood as well as in suspensions of platelets and red blood cells reconstituted in plasma. The by the cyclooxygenase inhibitor, indomethacin, and the thromboxane A2-receptor blocker, SQ30741, abolished the stimulation by TPO. The effect of TPO was mimicked by a very low concentration (10 nmol/l) of the thromboxane TxA2 analogue, U46619. Real-time studies of platelet adhesion to a VWF-coated surface at a shear of 1000/s showed that about 20% of the platelets were in a rolling phase before they became firmly attached. TPO (1 ng/ml) pretreatment reduced this number to < 5%, an effect again abolished by indomethacin. Thus, TPO potentiates the direct and firm attachment of platelets to surface-coated ligands for alphaIIbbeta3, possibly by increasing the ligand affinity of the integrin.

Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.