How Does Heparin Transform Pf4 Into An Alloantigen

PF4 is a 7.8 kDa CXC chemokine protein present in platelet a-granules as a tetramer of about 30 kDa. Upon platelet activation or lysis, PF4 is released into blood as a high molecular weight complex (350 kDa) consisting of a proteoglycan dimer carrying eight PF4 tetramers. PF4 is rapidly cleared from blood through binding to EC glycosaminoglycans (GAGs), for which it has a greater affinity than for the platelet proteoglycan dimer. During heparin therapy (UFH or LMWH), PF4 tetramers are displaced from the EC storage sites (because heparins have a higher affinity for PF4 than do other GAGs) and they are released into blood at concentrations that depend on the patient's platelet activation status (Fig. 1). In some cases, these PF4-H complexes induce the generation of heparin-dependent antibodies (Poncz, 2005).

Antibodies to self-antigens, including certain autologous plasma proteins, can develop as a result of immune dysfunction, triggering autoimmune disease. Sometimes, however, formation of complexes between an autologous protein and a foreign substance leads to new antigens on the self protein, which can be described as cryptic alloantigens or neoantigens. Figure 2 shows how the PF4 tetramer can be modified by its binding to heparin, thereby exposing neoepitopes that were masked on native PF4. The immune stimulation resulting from such an altered self-epitope abates quickly once the inducing foreign substance is no longer present. Such a model explains some of the clinical events observed in HIT (see Chapter 2). In HIT, PF4 constitutes the self antigen, forming an "alloantigen" when complexed with heparin, particularly when both PF4 and heparin are present at the stoichiometric concentrations that allow formation of multimolecular

FIGURE 1 (See color insert) Release of PF4 from platelets as a high molecular weight complex of eight tetramers with a proteoglycan dimer; PF4 binds to endothelial cell GAGs, for which it has a greater affinity, but it is displaced by heparin, which exhibits a higher affinity for PF4. Abbreviations: GAGs, glycosaminoglycans; PF4, platelet factor 4; PF4-H, PF4 complexed to heparin.

FIGURE 1 (See color insert) Release of PF4 from platelets as a high molecular weight complex of eight tetramers with a proteoglycan dimer; PF4 binds to endothelial cell GAGs, for which it has a greater affinity, but it is displaced by heparin, which exhibits a higher affinity for PF4. Abbreviations: GAGs, glycosaminoglycans; PF4, platelet factor 4; PF4-H, PF4 complexed to heparin.

complexes and, consequently, exposure of one or more cryptic neoepitopes. Thus, the antibodies to PF4-H complexes essentially can be considered to be autoantibodies or alloantibodies (Shoenfeld, 1997). However, as a foreign substance (heparin) is involved, and as there is little evidence that complexes between PF4 and naturally occurring GAGs generate antibodies, it seems more appropriate to call those heparin-dependent antibodies, "alloantibodies."

As mentioned, PF4 is a positively charged tetrameric glycoprotein (GP) member of the CXC chemokine family (Brandt and Flad, 1992). The tetramer forms by sequential noncovalent association of identical PF4 monomers: two dimers are formed that self-associate into the fundamental tetrameric structure. As found within platelet a-granules, PF4 is released into blood only after platelet activation, such as seen with trauma, surgery, atherosclerosis (Dunlop et al., 1987), diabetes, CPB, inflammation, cancer, infections, and so on. In vivo, PF4 has many different biological functions, including immunoregulation, inhibition of megakaryocytopoi-esis and angiogenesis, and mediation of cell response (Nesmelova et al., 2005; Slungaard, 2005). As summarized in Figure 1, PF4 released from platelets is in a 350 kDa complex comprised of eight PF4 tetramers linked to a chondroitin-contain-ing proteoglycan dimer (Barber et al., 1972; Luscombe et al., 1981; Huang et al., 1982). These PF4 complexes then bind to EC proteoglycans (heparan sulfate), and heparin, when present, displaces PF4 from the EC GAGs due to its greater affinity for PF4. The resulting PF4-H complexes are released into the circulating blood.

The interaction between heparin and PF4 has been intensively studied (Bock et al., 1980; Cowan et al., 1986; Stuckey et al., 1992; Maccarana and Lindahl, 1993). In the presence of a stoichiometric concentration of heparin and PF4 (which corresponds to about 27 international units [IU], i.e., about 175 ± 25 mg of heparin per milligram of PF4), multimolecular PF4-H complexes are generated (Greinacher et al., 1994; Amiral et al., 1995). With stoichiometric concentrations, heparin tightly wraps around the PF4 molecule (Fig. 2a), altering its structure and rendering it antigenic through the exposure of neoantigens. Figure 2b shows the different

PF4 tetramers

Heparin Binding Site Pf4

PF4 excess (B) (No complexes)

Stoichiometric PF4-Heparin (Formation of multimolecular complexes)

FIGURE 2 (See color insert) Schema showing the "modifications" of the PF4 tetramer following its tight binding with heparin at stoichiometry and exposure of neoepitopes (A) and depicting the formation of heparin and PF4 complexes at different concentrations of heparin and PF4 (B). In the presence of stoichiometric concentrations of both substances, multimolecular complexes are formed. Heparin then wraps around the PF4 tetramer, altering its structure and rendering it antigenic. Abbreviation: PF4, platelet factor 4.

PF4 tetramers

Heparin excess (No complexes)

PF4 excess (B) (No complexes)

Stoichiometric PF4-Heparin (Formation of multimolecular complexes)

Heparin excess (No complexes)

FIGURE 2 (See color insert) Schema showing the "modifications" of the PF4 tetramer following its tight binding with heparin at stoichiometry and exposure of neoepitopes (A) and depicting the formation of heparin and PF4 complexes at different concentrations of heparin and PF4 (B). In the presence of stoichiometric concentrations of both substances, multimolecular complexes are formed. Heparin then wraps around the PF4 tetramer, altering its structure and rendering it antigenic. Abbreviation: PF4, platelet factor 4.

complexes that can be formed between heparin and PF4, depending on the respective concentrations of both substances. Only multimolecular complexes, formed when well-defined ratios between PF4 and heparin exist, are believed to be antigenic. The larger PF4-H complexes formed with UFH, compared with LMWH, are believed to be more immunogenic and more pathogenic (Poncz, 2005; Rauova et al., 2005; Greinacher, 2006). Since complex formation depends strictly on the respective concentrations of heparin and PF4, if we consider the usual therapeutic range for heparin to be 0.1-1 IU/mL, then the amount of PF4 required for the generation of multimolecular PF4-H complexes ranges from 3 to 40 mg/mL. In patients undergoing CPB, who receive higher heparin concentrations (up to 3IU/ mL), the correspondingly higher PF4 concentrations required to form immunogenic PF4-H complexes results from the intense platelet activation produced when blood is exposed to the CPB circuit. In general, the existence of favorable conditions permitting formation of multimolecular PF4-H complexes may depend as much upon the underlying disease promoting platelet activation as on the dose of heparin given (see Chapter 3). Indeed, PF4 can be present at very high concentrations (exceeding the expected serum concentration of about 5 mg/mL) at pathological sites where platelets and leukocytes are chemoattracted and then activated (Fig. 3), such as during major surgery (orthopedic, cardiac), acute infection or inflammation, malignancy, etc.

Endothelial cells

Endothelial cells

Heparin Endothelial Cells

Prolonged immobilization inducing neutrophil (Neutr) and platelet (Plt) activation

FIGURE 3 (See color insert) Cell-cell interactions in the neighborhood of blood activation or inflammation sites: Presence of heparin-dependent antibodies increases the amount of cells available at these sites, amplifies cell-cell interactions and cellular activation, and can lead to blood clotting or release of circulating cell aggregates. The procoagulant effect is enhanced by release of tissue factor (from endothelial cells and monocytes) and generation of microparticles. Abbreviations: IL-8, interleukin-8; PF4, platelet factor 4; PF4-H, platelet factor 4-heparin complex; TF, tissue factor.

Prolonged immobilization inducing neutrophil (Neutr) and platelet (Plt) activation

FIGURE 3 (See color insert) Cell-cell interactions in the neighborhood of blood activation or inflammation sites: Presence of heparin-dependent antibodies increases the amount of cells available at these sites, amplifies cell-cell interactions and cellular activation, and can lead to blood clotting or release of circulating cell aggregates. The procoagulant effect is enhanced by release of tissue factor (from endothelial cells and monocytes) and generation of microparticles. Abbreviations: IL-8, interleukin-8; PF4, platelet factor 4; PF4-H, platelet factor 4-heparin complex; TF, tissue factor.

The intensity of the heparin-dependent immune response thus depends on the presence and, probably, the persistence of multimolecular PF4-H complexes. In particular, presence of high concentrations of PF4-H complexes may be important in triggering an immune response. However, heparin concentrations can vary considerably even in an individual patient during the course of heparin therapy, and so the stoichiometric concentrations allowing for PF4-H complex formation may occur frequently. But, if only low PF4 concentrations are present, formation of immunogenic complexes occurs only at corresponding low levels of heparin, e.g., 0.027IU/mL of heparin for 100ng/mL of PF4, which is the approximate highest PF4 concentration in normal subjects receiving heparin. The chances of developing a significant immune response in this setting would be low. In contrast, PF4 concentrations are expected to be high at sites where platelets and leukocytes converge and are activated (Fig. 3), thus improving the chances of an immune response. The potentially important role of individual immune responsiveness to a given PF4-H antigenic stimulus is unknown. Recent studies have demonstrated that the immunological response to a PF4-H immunological stimulus is T-cell dependent (Bacsi et al., 1999; Suvarna et al., 2005).

We have reported that although antibodies to PF4-H complexes are present in most patients who develop HIT, they are absent in some patients with apparent HIT, including patients with positive platelet activation assays. Antibodies to IL-8 or to NAP-2 have been observed in some of these patients (Amiral et al., 1996a; Regnault et al., 2003), but in a few, no specific heparin-dependent antibodies have yet been identified. As discussed later, antibodies to IL-8 or to NAP-2 can be generated by mechanisms different from those involved in PF4-H antibody formation. These antibodies can predate the heparin therapy, and could have a regulatory role for inflammation (Reitamo et al., 1993). Administration of heparin is then the trigger for their pathologic effect, as heparin then promotes binding of these chemokines onto some blood cells (including platelets and ECs) to which they do not bind (or bind poorly) in the absence of heparin. In contrast to anti-PF4-H antibodies, these anti-IL-8 or anti-NAP-2 antibodies might be true autoantibodies (Bendtzen et al., 1995). They can be generated in many different clinical situations, and their pathological incidence still remains unknown.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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