Antigen Generation in Heparin-Associated Thrombocytopenia: The Nonimmunologic Type and the Immunologic Type Are Closely Linked in Their Pathogenesis

Greinacher, Andreas

doi:10.1055/s-2007-1000384

Cited by 161 publications

(65 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It is well known that a critical heparin chain length of ;12 saccharide units is required to form PF4/polyanion complexes that express the antigen to which anti-PF4/heparin antibodies bind 28 and induce subsequent platelet activation. 24 10-mer heparin fragments induce only weak recognition, and 8-and 6-mer heparin fragments are even less 29,30 or nonreactive. 28 Consistent with these findings, anti-PF4/heparin antibodies did not (or only minimally) bind to PF4/HO08 and PF4/HO06 complexes in our study.…”

Section: Discussionmentioning

confidence: 98%

“…This excellently matches with empirically observed interaction patterns of PF4 with different heparins showing that $12 monosaccharides are necessary to induce anti-PF4/heparin antibody binding in vitro. [28][29][30] This critical heparin chain length can therefore also be interpreted by the minimum chain length that is necessary to release enough energy to drive the PF4 conformational changes, which finally lead to epitope exposure. 16 The CD experiments showed different patterns of the antiparallel b-sheet content of PF4 (Figure 1), which was reversible at higher concentrations when UFH, LMWH, and HO16 were added, but irreversible with HO08 and HO06.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Binding of anti–platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4

Kreimann¹,

Brandt²,

Krauel³

et al. 2014

Blood

Self Cite

View full text Add to dashboard Cite

Key Points• Besides clustering, platelet factor 4/polyanion complexes require input of energy to become immunogenic. • Minute differences in chain length determine the induction of antigenicity of PF4.The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel b-sheets exceeding ∼30% (achieved by UFH, LMWH,, (2) formation of multimolecular complexes (UFH,, and (3) energy (needed for a conformational change), which is released by binding of ‡11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic. (Blood. 2014;124(15):2442-2449 IntroductionHeparin, widely used clinically as a parenteral anticoagulant, 1-3 is a polyanion consisting of iduronic acid, glucuronic acid, and glucosamine residues carrying sulfate groups. Pharmaceutical heparin is obtained from porcine gut mucosa and, as biological material, is composed of polysaccharide chains with variable length. Degradation of unfractionated heparin (UFH) results in less polydisperse and smaller low-molecular-weight heparins (LMWHs). The pentasaccharide fondaparinux consists of the shortest sequence able to catalyze the activity of antithrombin. It was the first synthesized heparin approved for clinical use. 4 Currently, several other heparin-based, synthesized polysaccharides are in preclinical development. 5,6 Beside their anticoagulant activity, heparins have other biological effects including potential antitumor activity, 7 which, however, differs depending on the chain length. Clinically, beside bleeding, heparin-induced thrombocytopenia (HIT) is the most important adverse effect of heparin.8 HIT is a lifethreatening immune-driven adverse effect, which occurs in up to 3% of patients receiving UFH after major surgery. 9 HIT is caused by antibodies that recognize platelet factor 4 (PF4), a CXC chemokine family protein, in ultralarge multimolecular complexes with heparin. 10Several of these pathogenic antib...

show abstract

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Binding of anti–platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4

Kreimann¹,

Brandt²,

Krauel³

et al. 2014

Blood

Self Cite

View full text Add to dashboard Cite

show abstract

“…When blood comes into contact with the membrane, platelets adhere, aggregate and form a plug that occludes the aperture and stops the blood flow. The time required to occlude the aperture is automatically reported as the closure time (CT) [27, 28]. …”

Section: Methodsmentioning

confidence: 99%

Statin Potentiates Human Platelet eNOS Activity without Enhancing eNOS mRNA and Protein Levels

Yemişçi

Kocaefe³

et al. 2008

Cerebrovasc Dis

View full text Add to dashboard Cite

Background/Aims: Experimental studies suggest an enhanced endothelial and platelet nitric oxide (NO) generation after statin treatment, possibly due to increased endothelial NO synthase (eNOS) activity and protein levels. In parallel with experimental research, statins were shown to increase the forearm blood flow independently of serum cholesterol in humans. However, it was not possible to correlate blood flow changes with eNOS levels in these studies due to limitations in obtaining arterial samples. Hence, we investigated changes in eNOS activity, mRNA and protein levels after statin treatment in human platelets, which are readily accessible unlike arteries. Methods: In vitro bleeding times were measured in 22 patients by stimulating platelets with collagen-epinephrine or collagen-ADP. To assess platelet eNOS activity, the bleeding times were also determined after incubating platelets with L-arginine. The measurements were repeated following 14 days of pravastatin (40 mg/day) treatment. Platelet-rich plasma was collected before and after statin treatment to evaluate eNOS mRNA (semiquantitative RT-PCR) and protein levels (Western blotting). Results: The basal bleeding time was prolonged by 24 ± 3% (mean ± SE) when the samples were incubated with 500 µM of L-arginine. The NOS inhibitor L-N⁵-(I-iminoethyl)ornithine reversed this effect, suggesting that it was mediated by NO. After statin treatment, the NO-mediated prolongation of the bleeding time with 500 µM of L-arginine was significantly potentiated (to 44 ± 10%). Despite enhanced eNOS activity, there was no significant change in platelet eNOS mRNA and protein levels after statin treatment. Conclusion: These data demonstrate that platelet eNOS activity is potentiated after statin treatment in humans in parallel with experimental studies.

show abstract

“…3,4 These so-called nonimmune effects are much more moderate than the kind of platelet activation events that are precipitated by the binding to platelet Fc␥RIIa of patient antibodies that react with platelet factor 4 (PF4)/heparin complexes in the disorder heparin-induced thrombocytopenia (HIT), 5,6 although heparin binding-mediated platelet activation has been suggested, via secretion of platelet ␣-granule localized PF4, to play a role in the pathogenesis of immune HIT. 7 Although the precise mechanism by which heparin potentiates platelet activation in the absence of antibody/heparin/PF4 complexes is not well understood, there are numerous reports that heparin associates directly with the platelet surface, 8,9 produces minor, 10 though not always observed, 11 changes in platelet morphology, promotes a low level of P-selectin expression, 4 as well as the binding of fibrinogen 12 or the fibrinogen-mimetic antibody PAC-1, 4 to the major platelet integrin, ␣IIb␤3. Similar to other forms of agonist-induced platelet aggregation, heparin-potentiated platelet aggregation requires ␣IIb␤3, as it can be blocked with anti-␣IIb␤3-specific monoclonal antibodies, and does not occur in platelets from patients missing this integrin receptor.…”

Section: Introductionmentioning

confidence: 99%

Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling

Gao

Boylan

Fang

et al. 2011

Blood

113

View full text Add to dashboard Cite

Unfractionated heparin (UFH) is a widely used anticoagulant that has long been known to potentiate platelet responses to subthreshold doses of platelet agonists. UFH has been reported to bind and induce modest conformational changes in the major platelet integrin, ␣IIb␤3, and induce minor changes in platelet morphology. The mechanism by which UFH elicits these platelet-activating effects, however, is not well understood. We found that both human and murine platelets exposed to UFH, either in solution or immobilized onto artificial surfaces, underwent biochemical and morphologic changes indicative of a potentiated state, including phosphorylation of key cytosolic signaling molecules and cytoskeletal changes leading to cell spreading. Low molecular weight heparin and the synthetic pentasaccharide, fondaparinux, had similar plateletpotentiating effects. Human or mouse platelets lacking functional integrin ␣IIb␤3 complexes and human platelets pretreated with the fibrinogen receptor antagonists eptifibatide or abciximab failed to become potentiated by heparin, demonstrating that heparin promotes platelet responsiveness via its ability to initiate ␣IIb␤3-mediated outside-in signaling. Taken together, these data provide novel insights into the mechanism by which platelets become activated after exposure to heparin and heparin-coated surfaces, and suggest that currently used glycoprotein IIb-IIIa inhibitors may be effective inhibitors of nonimmune forms of heparin-induced platelet activation.

show abstract

Antigen Generation in Heparin-Associated Thrombocytopenia: The Nonimmunologic Type and the Immunologic Type Are Closely Linked in Their Pathogenesis

Cited by 161 publications

References 64 publications

Binding of anti–platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4

Binding of anti–platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4

Statin Potentiates Human Platelet eNOS Activity without Enhancing eNOS mRNA and Protein Levels

Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling

Contact Info

Product

Resources

About