The procoagulant effects of air pollution.
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Air Pollution↗
Biomedical subjects
M F Hoylaerts
Publications and source records attributed to M F Hoylaerts.
Flow-based assays for global assessment of hemostasis. Part 1: Biorheologic considerations.
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Blood Coagulation↗
Flow-based assays for global assessment of hemostasis. Part 2: current methods and considerations for the future.
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Blood Coagulation Disorders↗
von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen.
BACKGROUND: Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively. OBJECTIVES: Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen. METHODS AND RESULTS: In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-A3 both competed for binding of DeltaA1-VWF and DeltaA3-VWF to horse tendon collagen fibrils in static conditions and to human collagen III during plasmon surface resonance studies, substantiating overlapping binding sites on both collagens for A1 and A3. Heparin did not affect A3-mediated binding of VWF and DeltaA1-VWF, but inhibited binding to horse tendon collagen of GST-A1 and DeltaA3-VWF. Furthermore, A1-mediated binding to type III collagen of DeltaA3-VWF binding was strongly salt-sensitive. During perfusions at wall shear rate 2500 s(-1) of calcein-labeled platelets in reconstituted blood, DeltaA3-VWF and H1786A-VWF triggered platelet binding to horse tendon collagen comparably and as potently as VWF, and to human type III collagen, only fivefold less potently, DeltaA1-VWF being inactive. Additional flow-controlled interaction studies with DeltaA3-VWF, H1786A-VWF, the collagen-VWF antagonist saratin, heparin and the VWF neutralizing antibody 82D6A3 confirmed that H1786A-VWF binds to collagen exclusively via A1. CONCLUSION: Hence, in shear forces the VWF A1 domain can assume the role of A3 to trigger substantial platelet recruitment to human collagen fibres.
Animals↗
Haemostasis.
When the continuity of the vascular endothelium is disrupted, platelets and fibrin seal off the defect. Haemostatic processes are classified as primary (mainly involving platelets) and secondary (mainly related to fibrin formation or blood coagulation). When the blood clot is no longer required for haemostasis, the fibrinolytic system will dissolve it. The pivotal ligand for initial platelet recruitment to injured vessel wall components is von Willebrand factor (vWF), a multimeric protein present in the subendothelium and in plasma, where it is conformationally activated by shear forces. Adhering activated platelets recruit additional platelets, which are in turn activated and form a platelet aggregate. Coagulation is initiated by a reaction, activating factors IX and X. Once critical amounts of factor Xa are generated, thrombin generation is initiated and soluble fibrinogen is converted into insoluble fibrin. Excessive thrombin generation is prevented via inhibition by antithrombin and also via downregulation of its further generation by activation of the protein C pathway. Activation of the fibrinolytic system results from conversion of the proenzyme plasminogen into the active serine proteinase plasmin by tissue-type or urokinase-type plasminogen activators. Plasmin digests the fibrin component of a blood clot. Inhibition of the fibrinolytic system occurs at the level of the plasminogen activator (by plasminogen activator inhibitors) or at the level of plasmin (by alpha2-antiplasmin). Together, these physiological processes act to maintain normal functioning blood vessels and a non-thrombotic state.
Animals↗
ERK2 activation in arteriolar and venular murine thrombosis: platelet receptor GPIb vs. P2X.
The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). In SIPA, ERK2 was rapidly phosphorylated during GPIb stimulation, its activation contributing to SIPA for 50%, independently of P2X(1) regulation. Thrombotic occlusion of injured arterioles occurred considerably faster in TG (4.3 +/- 2.3 min) than in WT (38 +/- 8 min) arterioles, but occlusion times in TG (19 +/- 12) and WT (48 +/- 4.5 min) venules differed less. Both the alphabeta-meATP triggered desensitization of platelet P2X(1), as well as P2X(1) antagonism by NF279 or NF449 prolonged mean occlusion to about 75 min in WT and 65 min in TG arterioles, but venular occlusion times were less affected. Preventing ERK2 activation by U0126 prolonged occlusion times in TG (41 +/- 10 min) and WT (51 +/- 17) arterioles more than in TG (46 +/- 5 min) and WT (56 +/- 6 min) venules, uncovering a role for ERK2 in shear controlled thrombosis. Antagonism of GPIb by a recombinant murine von Willebrand factor (VWF)-A1 fragment prolonged occlusion times to comparable values, ranging from 55 to 58 min, both in TG and WT arterioles and venules. Further inhibition strategies, combining VWF-A1, U0126 and NF449 in WT and TG mice and resulting in occlusion in various time windows, identified that inhibition by VWF-A1 largely abrogated the ERK2 contribution to thrombosis. In conclusion, P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions.
Animals↗
The platelet ATP and ADP receptors.
Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.
Animals↗
Ambient air pollution and acute myocardial infarction.
This review summarizes the nature of ambient air pollutants, which are either gaseous or particulate of various sizes, the latter determining their penetration into the body, the smallest even translocating from the lung into the systemic circulation. It presents the epidemiological evidence linking air pollution to overall mortality, cardiovascular mortality and myocardial infarction, making the distinction between acute and chronic exposure to the pollutants. It reviews mechanistic investigations that have evaluated the links among exposure to pollutants, thrombosis, pulmonary inflammation, arterial vasoconstriction and heart rate variability. It concludes by attempting to integrate current epidemiological and mechanistic observations into a pathophysiological framework that links ambient air pollution to acute myocardial infarction and cardiovascular mortality.
Air Pollution↗
alphaIIbbeta3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium.
Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antialpha(IIb)beta(3) antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that alpha(IIb)beta(3) antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s(-1), largely VWF-independent. When perfused over a human umbilical vein endothelial cell-derived extracellular matrix, single alpha(IIb)beta(3)-antagonized platelets primarily adhered to matrix-bound VWF when perfused at 2700 s(-1), but at 1300 s(-1) they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de-endothelialized rabbit aorta at a wall shear rate of 1100 s(-1), alpha(IIb)beta(3) antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected alpha(IIb)beta(3)-antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, alpha(IIb)beta(3) antagonism inhibited platelet-rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than alpha(IIb)beta(3) antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.
Animals↗
Do lipid rafts contribute to platelet activation?
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Blood Platelets↗
Passage of inhaled particles into the blood circulation in humans.
BACKGROUND: Pollution by particulates has been consistently associated with increased cardiovascular morbidity and mortality. However, the mechanisms responsible for these effects are not well-elucidated. METHODS AND RESULTS: To assess to what extent and how rapidly inhaled pollutant particles pass into the systemic circulation, we measured, in 5 healthy volunteers, the distribution of radioactivity after the inhalation of "Technegas," an aerosol consisting mainly of ultrafine (99m)Technetium-labeled carbon particles (<100 nm). Radioactivity was detected in blood already at 1 minute, reached a maximum between 10 and 20 minutes, and remained at this level up to 60 minutes. Thin layer chromatography of blood showed that in addition to a species corresponding to oxidized (99m)Tc, ie, pertechnetate, there was also a species corresponding to particle-bound (99m)Tc. Gamma camera images showed substantial radioactivity over the liver and other areas of the body. CONCLUSIONS: We conclude that inhaled (99m)Tc-labeled ultrafine carbon particles pass rapidly into the systemic circulation, and this process could account for the well-established, but poorly understood, extrapulmonary effects of air pollution.
Adult↗
Passage of intratracheally instilled ultrafine particles from the lung into the systemic circulation in hamster.
The mechanisms of particulate pollution-related cardiovascular morbidity and mortality are not well understood. We studied the passage of radioactively labeled ultrafine particles after their intratracheal instillation. Hamsters received a single intratracheal instillation of 100 microg albumin nanocolloid particles (nominal diameter < or = 80 nm) labeled with 100 microCi technetium-99m and were killed after 5, 15, 30, and 60 min. In blood, radioactivity, expressed as percentage of total body radioactivity per gram blood, amounted to 2.88 +/- 0.80%, 1.30 +/- 0.17%, 1.52 +/- 0.46%, and 0.21 +/- 0.06% at 5, 15, 30, and 60 min, respectively. Thin-layer chromatography showed only one peak of radioactivity corresponding to unaltered (99m)Tc-albumin nanocolloid. In the liver, radioactivity, expressed as percentage of total radioactivity per organ, amounted to 0.10 +/- 0.07%, 0.23 +/- 0.06%, 1.24 +/- 0.27%, and 0.06 +/- 0.02% at 5, 15, 30, and 60 min, respectively. Lower values were observed in the heart, spleen, kidneys, and brain. Dose dependence was assessed at 30 min following instillation of 10 microg and 1 microg (99m)Tc-albumin per animal (n = 3 at each dose), and values of the same relative magnitudes as after instillation of 100 microg were obtained. We conclude that a significant fraction of (99m)Tc-albumin, taken as a model of ultrafine particles, rapidly diffuses from the lungs into the systemic circulation.
Air Pollutants↗
Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1 mutation.
A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for beta-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbbeta and GPIX but also a significantly lower expression of the nondirectly GATA1-regulated Gsalpha gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbalpha, GPIbbeta, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion, GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
Adult↗
Identification of a target site in plasminogen activator inhibitor-1 that allows neutralization of its inhibitor properties concomitant with an allosteric up-regulation of its antiadhesive properties.
The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (MA-124K1) that inhibits rat PAI-1 activity and simultaneously increases the binding of inactive PAI-1 to vitronectin (the affinity constant of PAI-1 for vitronectin is 2 x 10(7) m(-1) in the absence of MA-124K1 and 160 x 10(7) m(-1) in the presence of MA-124K1). To the best of our knowledge, this is the first monoclonal antibody dissociating the proteinase inhibitory properties from the vitronectin binding properties in PAI-1. Mutation of Glu(212) and/or Glu(220) in rat PAI-1 to Ala results in a strongly reduced affinity or absence of binding to MA-124K1. The three-dimensional structure of PAI-1 reveals that these residues constitute a conformational epitope close to the reactive-site loop and compatible with the effect of MA-124K1 on the inhibitory properties of PAI-1. However, the vitronectin binding site is localized at the opposite site of the molecule, indicating that the effect of MA-124K1 involves an allosteric modulation of the vitronectin binding site. Cell culture experiments revealed a significant reduction of cell attachment and migration in the presence of MA-124K1, providing evidence for the functional relevance of this antibody-mediated up-regulation of the vitronectin binding properties of PAI-1. In conclusion, a novel mechanism for interference with PAI-1 functions has been identified and is of importance in the modulation of cell migration and related events (e.g. tumor metastasis).
Allosteric Regulation↗
Mouse carotid artery ligation induces platelet-leukocyte-dependent luminal fibrin, required for neointima development.
The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor-positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28+/-3.7% (n=11) and 42+/-5.1% (n=8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII(-/-) mice, stenosis was significantly lower 1 (11+/-3.6%, n=8) and 2 (21+/-4.7%, n=7) weeks after ligation (both P:<0.01 versus background-matched controls). In u-PA(-/-) mice, luminal stenosis was significantly higher 1 (38+/-7.0%, n=7) and 2 (77+/-5.6%, n=6) weeks after ligation (P:<0.05 and P:<0.01, respectively, versus matched controls). In alpha(2)-AP(-/-) mice, stenosis was lower at 1 week (14+/-2.6%, n=7, P:<0.01) but not at 2 weeks. Responses in tissue-type plasminogen activator or plasminogen activator inhibitor-1 gene-deficient mice equaled that in controls. Reducing plasma fibrinogen levels in controls with ancrod or inducing partial thrombocytopenia with busulfan resulted in significantly less neointima, but inflammation was inhibited only in busulfan-treated mice. We conclude that stasis induces platelet activation, leading to microthrombosis and platelet-leukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which is used by smooth muscle cells to migrate into the lumen.
Afibrinogenemia↗
Congenital disorders of glycosylation type Ia and IIa are associated with different primary haemostatic complications.
Congenital disorders of glycosylation (CDG) type I are mostly due to a deficient phosphomannomutase activity, called CDG Ia. CDG IIa (mutations in the MGAT2 gene) results from a deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II. CDG Ia patients predominantly have a thrombotic tendency, whereas our CDG IIa patient has an increased bleeding tendency, despite similar coagulation factor abnormalities in both types. We have investigated whether abnormally glycosylated platelet membrane glycoproteins are involved in the haemostatic complications of both CDG groups. In flow cytometry, the binding of Ricinus communis lectin (reactive with beta-galactose primarily) to control platelets increased after neuraminidase treatment: this increase was smaller (p < 0.01) in CDG Ia patients (3.1 +/- 0.08 times) than in control platelets (8.5 +/- 1.8 times) and did not occur in the CDG IIa patient. Platelet-rich plasma from CDG Ia patients, but not a CDG IIa patient. aggregated spontaneously and gel-filtered platelets from CDG Ia patients agglutinated at very low concentrations of ristocetin, independently of von Willebrand factor (vWF). Accordingly, in stirred whole blood, the rate of single platelet disappearance of CDG Ia patients was twice that of control platelets. In contrast, perfusion of whole anticoagulated blood of the CDG IIa patient over collagen yielded markedly decreased platelet adherence to collagen at shear rates involving glycoprotein (GP) Ib-vWF interactions. Thus, abnormal glycosylation of platelet glycoproteins in CDG Ia enhances nonspecific platelet interactions, in agreement with a thrombotic tendency. The reduced GP Ib-mediated platelet reactivity with vessel wall components in the CDG IIa patient under flow conditions provides a basis for his bleeding tendency.
Blood Platelets↗
Anti-vWf antibodies induce GPIbalpha and FcgammaRII mediated platelet aggregation only at low shear forces.
BACKGROUND/METHODS: Anti-von Willebrand factor (vWf) antibody mediated platelet activation was studied using 2 monoclonal anti-vWf antibodies promoting the binding of vWf to GPIbalpha: 1C1E7 (IgG2a) reacting with the vWf N-terminus and 75H4B12 (IgM), characterized in this paper and studied in association with 1C1E7. RESULTS: 75H4B12 binds to an N-terminal epitope in vWf, different from that reacting with 1C1E7. When com-bined, 1C1E7 and 75H4B12 promoted vWf binding to isolated GPIb under static conditions, even in the absence of ristocetin or botrocetin, and induced platelet aggregation synergistically in the presence of zero to subthreshold ristocetin concentrations. Specific inhibitors of GPIbalpha-vWf interactions prevented vWf binding to GPIbalpha in ELISA and during platelet aggregation. In addition, the 1C1E7 dependent platelet aggregation involved Fc receptor mediated platelet activation, a phenomenon even more pronounced when 1C1E7 and 75H4B12 were combined. A 75H4B12 binding phage expressing a peptide homologous with vWf sequence 88-95 neutralized the antibody induced platelet activation. However, at arterial shear rates, both 1C1E7 and 75H4B12 potently prolonged cartridge closure times in the PFA-100, compatible with inhibition of platelets by vWf, unfolded by the combined action of shear stress and antibodies. CONCLUSIONS: We conclude that antibodies directed against different epitopes in the N-terminus of vWf modify the folded vWf structure synergistically and enhance A1 domain mediated vWf binding to platelet GPIb at low shear forces. In addition, once platelet-bound, IgG antibodies potently activate platelets via the FcgammaII receptor. Thus, such antibodies may promote immune mediated thrombosis at low shear rates, typical of the venous circulation. In contrast, at arterial shear rates, anti-vWf antibodies may rather compromise platelet function following enhanced binding of the unfolded vWf multimers to platelets, shielding platelets from interacting with subendothelial and soluble ligands.
Antibodies, Monoclonal↗
Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen.
Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.
Animals↗