Perumal Thiagarajan

P-Selectin Expression on Platelets Determines Size and Stability of Platelet Aggregates

BackgroundP-selectin mediates rolling of platelets and leukocytes on activated endothelial cells. After platelet activation, P-selectin is translocated from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein (GP) IIb/IIIa between adjacent platelets. Methods and ResultsIn this study, we define a novel role for P-selectin in platelet aggregation. Expression of P-selectin on the platelet surface correlated strongly with the mean platelet aggregate size. Inhibition of P-selectin binding to its ligand by either monoclonal anti–P-selectin antibodies directed against the lectin domain or soluble human P-selectin reversed platelet aggregation even when added up to 5 minutes after activation; however, fibrinogen binding to platelets was not affected. This deaggregating effect significantly reduced the maximal size and number of platelet aggregates. When added 1 minute after platelet activation, anti–P-selectin antibody achieved 95% to 100% of the deaggregating effect of EDTA, whereas the anti-GP IIb/IIIa antibody abciximab had no effect. Monoclonal antibodies against known P-selectin ligands, such as P-selectin GP ligand-1 (PSGL-1) or GP Ib, had no effect on platelet aggregation, suggesting a different ligand for P-selectin in platelet aggregate stabilization. In kinetic studies, P-selectin was maximally expressed 10 minutes after platelet activation, whereas maximal activation of GP IIb/IIIa occurred within the first 10 seconds, suggesting that P-selectin operates after fibrinogen binding to activated GP IIb/IIIa. ConclusionsThese results indicate that P-selectin interaction with a ligand, different from PSGL-1 or GP Ib, stabilizes initial GP IIb/IIIa–fibrinogen interactions, allowing the formation of large stable platelet aggregates.

Platelet Microparticles Promote Platelet Interaction With Subendothelial Matrix in a Glycoprotein IIb/IIIa–Dependent Mechanism

BACKGROUNDnPlatelets, on activation, release vesicular particles called platelet microparticles. Despite their procoagulant activity, their functional role in platelet-vessel wall interactions is not known.nnnMETHODS AND RESULTSnWe examined the binding of microparticles to vessel wall components in vitro and in vivo. Microparticles bound to fibrinogen-, fibronectin-, and collagen-coated surfaces. Compared with activated platelets, we observed minimal binding of microparticles to vitronectin and von Willebrand factor. The glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors abciximab and eptifibatide (Integrilin) inhibited the binding to fibrinogen and fibronectin but had minimal effect on binding to collagen. Furthermore, monoclonal antibodies to GP Ib or anionic phospholipid-binding proteins (beta2-glycoprotein I or annexin V) had no effect in these interactions. Microparticles did not bind to monolayers of resting or stimulated human umbilical vein endothelial cells (HUVECs), even in the presence of fibrinogen or von Willebrand factor. However, under similar conditions, microparticles bound to extracellular matrix produced by cultured HUVECs. Abciximab inhibited this interaction by approximately 50%. In a rabbit model of arterial endothelial injury, the infusion of 51Cr-labeled microparticles resulted in a 3- to 5-fold increase of microparticle adhesion to the injured site compared with the uninjured site (P<0.05%). Furthermore, activated platelets bound to surface-immobilized microparticles in a GP IIb/IIIa-dependent mechanism. This binding increased in the presence of fibrinogen by approximately 30%.nnnCONCLUSIONSnPlatelet microparticles bind to subendothelial matrix in vitro and in vivo and can act as a substrate for further platelet binding. This interaction may play a significant role in platelet adhesion to the site of endothelial injury.

A New Role for P-Selectin in Shear-Induced Platelet Aggregation

BackgroundP-selectin, expressed on platelets on activation, mediates rolling of platelets on endothelial cells, but its role in shear-induced platelet aggregation is not known. Methods and ResultsPlatelets were exposed to either a single pulse (30 seconds) or 3 pulses (10 seconds) of high shear stress (150 to 200 dynes/cm2) each followed by low shear stress (10 dynes/cm2) for 4.5 minutes or 90 seconds, respectively, at 37°C to resemble more closely in vivo conditions such as those in stenotic arteries. Under these conditions, platelet aggregation was significantly increased compared with low or high shear stress alone. Monoclonal anti–P-selectin antibodies inhibited shear-induced platelet aggregation, especially when induced by the combination of high and low shear stress, by ≈70% and had an additive effect on the inhibition by abciximab (anti–glycoprotein (GP) IIb/IIIa antibody). However, anti–P-selectin antibody inhibited shear-induced platelet aggregation only at 37°C, not at 22°C, whereas abciximab inhibited shear-induced platelet aggregation at both 22°C and 37°C. This differential effect of anti–P-selectin antibody is explained by the finding that shear-induced P-selectin expression on platelets was observed mainly at 37°C. ConclusionsThese results indicate that pulsatile shear stress, which resembles flow conditions in stenotic arteries, induces significantly more platelet aggregation at 37°C than monophasic shear stress. Under these conditions, we show a novel role for P-selectin in platelet aggregation distinct from that of GP IIb/IIIa, which may be of importance in the initiation of thrombosis associated with atherosclerotic lesions.

Inhibition of Arterial Thrombosis by Recombinant Annexin V in a Rabbit Carotid Artery Injury Model

BACKGROUNDnThe procoagulant effect of anionic phospholipid may play a major role in the development of arterial thrombosis.nnnMETHODS AND RESULTSnAnnexin V, a calcium-dependent anionic-phospholipid-binding protein, was expressed and isolated from Escherichia coli and its antithrombotic effect examined in a rabbit carotid artery thrombosis model. A partially occlusive thrombus was formed in the left carotid artery by application of electric current to produce an approximately 50% occlusion of the lumen. After the current was discontinued, flow ceased completely within 42+/-12 minutes (n=6) because of continuing platelet/fibrin thrombus formation. When annexin V was given at doses of 2.8 to 16.6 microg x kg(-1) x min(-1) for a period of 180 minutes, starting at the time the current was stopped, there was a dose-dependent inhibition of thrombus formation. At a dose of 5.6 microg x kg(-1) x min(-1), blood flow remained patent throughout the infusion and for an additional 60 minutes after the infusion was stopped. In addition, there was a decrease in thrombus weight (16+/-7.4 versus 2.0+/-1.0 g), (125)I-fibrin deposition (approximately 45% reduction, P<.001), and (111)In-labeled platelet accumulation (approximately 43% reduction, P<.001). Prior mixing of annexin V with phosphatidylserine micelles abolished the antithrombotic effect of annexin V, whereas mixing with phosphatidylcholine micelles had no effect. The antithrombotic effect of annexin V was not associated with bleeding tendency, as judged by the amount of blood absorbed in a gauze pad placed in a surgical incision extending to the muscle tissue and by the standard template bleeding time.nnnCONCLUSIONSnThese observations support a potentially important role for anionic phospholipid exposure in platelets in arterial thrombosis, and inhibition of this activity could be a novel target for therapy in coronary thrombosis and stroke and after angioplasty.

Associations of anti-β2-glycoprotein I autoantibodies with HLA class II alleles in three ethnic groups

OBJECTIVEnTo determine any HLA associations with anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in a large, retrospectively studied, multiethnic group of 262 patients with primary antiphospholipid antibody syndrome (APS), systemic lupus erythematosus (SLE), or another connective tissue disease.nnnMETHODSnAnti-beta2GPI antibodies were detected in sera using an enzyme-linked immunosorbent assay. HLA class II alleles (DRB1, DQA1, and DQB1) were determined by DNA oligotyping.nnnRESULTSnThe HLA-DQB1*0302 (DQ8) allele, typically carried on HLA-DR4 haplotypes, was associated with anti-beta2GPI when compared with both anti-beta2GPI-negative SLE patients and ethnically matched normal controls, especially in Mexican Americans and, to a lesser extent, in whites. Similarly, when ethnic groups were combined, HLA-DQB1*0302, as well as HLA-DQB1*03 alleles overall (DQB1*0301, *0302, and *0303), were strongly correlated with anti-beta2GPI antibodies. The HLA-DR6 (DR13) haplotype DRB1*1302; DQB1*0604/5 was also significantly increased, primarily in blacks. HLA-DR7 was not significantly increased in any of these 3 ethnic groups, and HLA-DR53 (DRB4*0101) was increased in Mexican Americans only.nnnCONCLUSIONnCertain HLA class II haplotypes genetically influence the expression of antibodies to beta2GPI, an important autoimmune response in the APS, but there are variations in HLA associations among different ethnic groups.

Lupus anticoagulants and antiphospholipid antibodies

In recent years, clinical syndromes involving lupus anticoagulants and antiphospholipid antibodies have come into increasing clinical prominence. Since the discovery that most antiphospholipid antibodies require the presence of anionic phospholipid-binding proteins such as B2-glycoprotein I and prothrombin, a large number of studies have attempted to delineate the specificity of these antibodies. Several mechanisms have been proposed to explain the hypercoagulable state associated with these antibodies. This review attempts to summarize these data and the challenges that confront efforts to delineate the pathogenesis of the prothrombotic state associated with the presence of these antibodies.

Polymorphism of β2-glycoprotein I at codons 306 and 316 in patients with systemic lupus erythematosus and antiphospholipid syndrome

Objective n nTo determine the frequency of mutations in the phospholipid binding domain of β2-glycoprotein I (β2GPI) in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS), and to analyze the clinical correlations of such mutations with thromboembolic complications. n n n nMethods n nExons 7 and 8 of β2GPI, which encode for its fifth domain, were amplified by polymerase chain reaction, and the presence of mutations was determined by restriction digestion and single-strand conformation polymorphism analysis. A clinical correlation with these mutations and the presence of antiphospholipid antibodies (aPL), lupus anticoagulant (LAC), anti-β2GPI antibody, and the development of thromboembolic complications was performed using chi-square and Fishers exact tests. n n n nResults n nFrom a total of 143 patients studied, we found that 5.6% were heterozygous for the mutation at exon 7 (codon 306), and 7.7% were heterozygous for the mutation at exon 8 (codon 316). No homozygous subjects were found for either mutation. No significant correlation between these mutations and the presence of aPL, LAC, or anti-β2GPI antibodies was found. In patients with SLE (n = 95), 4 of 6 patients with exon 8 mutation had thrombosis, compared with 22 of 82 patients without the mutation (P = 0.043). n n n nConclusion n nThe prevalence of mutations in the fifth domain of β2GPI in these patients with SLE and/or APS were similar to those previously reported for the general population. Heterozygosity for either mutation does not influence the incidence of aPL, but in patients with SLE, the mutation at exon 8 may predispose to thrombosis as an independent factor.

Thrombin receptor activating peptide (SFLLRN) potentiates shear-induced platelet microvesiculation

Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles. In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec(-1) (shear stress approximately 375 dyne/cm2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation.

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library.

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.

Polymorphisms β2-glycoprotein. I: phospholipid binding and multimeric structure

beta2-glycoprotein I is a phospholipid-binding protein of 326 amino acids and is found in plasma at a concentration of approximately 200 microg/ml. It has a sequence of positively charged amino acids located at the carboxy terminus that mediates anionic phospholipid binding. Two polymorphisms (306Cys-->Gly and 316Trp-->Ser) located at the phospholipid-binding site have been described. Homozygous state for either mutation and a compound heterozygous state show no phospholipid binding. Interestingly, heterozygotes for either 306Cys-->Gly or 316Trp-->Ser mutation have normal cardiolipin binding suggesting that beta2-glycoprotein I may circulate as a multimeric structure where wild-type subunits compensate the defective binding of the mutant ones. We investigated the effect of these mutations on quaternary structure of beta2-glycoprotein I and phospholipid binding. As previously reported, under native conditions, beta2-glycoprotein I shows an apparent molecular weight of approximately 320 kDa and it can be dissociated into subunits of lower molecular weight by boiling in 6 M urea. We show that the multimeric structure is not affected by the presence of mutations in the phospholipid-binding domain. beta2-glycoprotein I induces aggregation of anionic phospholipid vesicles suggesting again a multivalent interaction where at least two binding sites are required to bridge adjacent vesicles. beta2-glycoprotein I-induced aggregation does not cause vesicle fusion or damage as demonstrated by fluorescence resonance energy transfer (FRET) or encapsulated calcein release. In conclusion, the normal cardiolipin binding in heterozygous state for mutations at phospholipid-binding domain may be due to the multimeric structure of beta2-glycoprotein I.