**3. Antibody-induced thrombocytopenia**

Immunocomplexes composed of aPF4/P Abs and PF4/polyanion (PF4/P) complexes on the platelet surface induce platelet aggregation via cross-linking FcγRIIA receptors [9, 10]. They also bind to the surface of endothelial cells and monocytes [44–46], inducing procoagulant activity [44, 47]. Heparin-induced thrombocytopenia has been well understood. Recent studies reported that a subset of human-derived autoantibodies in some patients also can induce thrombocytopenia in a heparin-similar manner.

#### **3.1. Human-derived HIT antibodies**

All aPF4/P Abs bind to immobilized PF4/P complexes in ELISA [48], but only some of them activate platelets in functional assays, e.g. the heparin-induced platelet activation assay (HIPA) [48] or the serotonin release assay (SRA) [49, 50]. Human-derived aPF4/P Abs compose of three groups, i.e. the antibodies do not activate platelets in HIPA test (group-1 Abs); the antibodies activate platelets in HIPA but require heparin (group-2 Abs); the antibodies activate platelets even without heparin (group-3 Abs) (**Figure 4**). Group-3 Abs developed from patients who had clinical autoimmune HIT, and therefore, they are defined as 'autoantibodies' [51].

**Figure 4.** Different reaction patterns of aPF4/H antibodies. (Right) pyramid shows antibodies of three groups, all positive in EIA. Group-1 (blue) do not activate platelets (HIPA -); many Abs belonging to group-2 do not induce HIT (yellow), some induce HIT (gold) and others induce HIT with thrombosis (dark red). Recent studies found an additional small subset of patient's content autoimmune group-3 HIT Abs (red). (Left) visualization of platelet aggregates-induced by different antibody groups imaged by scanning electron microscopy in the presence (+) or absence (−) of heparin: Group-1 abs induce (bottom left) only small aggregates reflecting the background platelet activation; group-2 Abs (middle, left) cause large aggregates only in the presence of heparin; group-3 Abs induce large aggregates even in the absence of heparin. Same scale bar for all images. Adapted from [55].

#### *3.1.1. Characteristics of human-derived HIT antibodies*

(i) PF4-Heparin and (ii) PF4-PF4 bond, whereas short heparins form only one PF4-Heparin bond. Even though the concept of the PF4-PF4 bond, in general, cannot be accepted because PF4s are highly positive proteins, and therefore, strongly repel each other. However, when forming a complex with a highly negative charged heparin, the positive-charged PF4 is probably neutralized that results in a mergence of two hydrophobic PF4 surfaces [34]. Based on these findings, a model for PF4-heparin interaction has been proposed (**Figure 3**). Due to their sizes, the short heparins simply bind to a single PF4 tetramer (**Figure 3A**), whereas the long heparins neutralize positive charges on PF4 tetramers and switch the charges between two PF4 tetramers from a repulsion to an attraction. Heparin reacts as a catalyst that forces two PF4 molecules close to each other within a distance l (l < L), resulting in two merged hydrophobic PF4 surfaces (**Figure 3B**). This way of interacting results in the extremely stable PF4/H

A sequence in the formation of PF4/heparin complexes has been identified. When a long heparin comes closely to PF4s, heparin forms first bonds with positively charged clusters on PF4s

Based on bond energy (ΔE), quantitative information of bond transitions can be calculated following the study of Wang et al. [43]. The bond transitions of short heparin from the weak positive-charged area on PF4 release energy, whereasPF4-PF4 bonds consume energy [35]. In contrast to short heparin, the bond transitions of long heparins in both cases release energy, while their interactions with the positively charged clusters consume energy (**Table 1**). Based on energy level, PF4-PF4 interaction is attributed to be stronger than the bonds between heparin and non-clusters of positive-charged areas on PF4. However, PF4-PF4 interaction is weaker than the interaction between heparin and clusters

Immunocomplexes composed of aPF4/P Abs and PF4/polyanion (PF4/P) complexes on the platelet surface induce platelet aggregation via cross-linking FcγRIIA receptors [9, 10]. They also bind to the surface of endothelial cells and monocytes [44–46], inducing procoagulant activity [44, 47]. Heparin-induced thrombocytopenia has been well understood. Recent studies reported that a subset of human-derived autoantibodies in some patients also can induce

All aPF4/P Abs bind to immobilized PF4/P complexes in ELISA [48], but only some of them activate platelets in functional assays, e.g. the heparin-induced platelet activation assay (HIPA) [48] or the serotonin release assay (SRA) [49, 50]. Human-derived aPF4/P Abs compose of three groups, i.e. the antibodies do not activate platelets in HIPA test (group-1 Abs); the antibodies activate platelets in HIPA but require heparin (group-2 Abs); the antibodies activate platelets even without heparin (group-3 Abs) (**Figure 4**). Group-3 Abs developed from patients who

had clinical autoimmune HIT, and therefore, they are defined as 'autoantibodies' [51].

and then it pulls closely PF4s together to form PF4-PF4 bonds [35].

complexes, especially for long heparins.

38 Thrombocytopenia

of positive charges on PF4.

**3. Antibody-induced thrombocytopenia**

thrombocytopenia in a heparin-similar manner.

**3.1. Human-derived HIT antibodies**

In contrast to the detailed characterization of the PF4/polyanion complexes, little is known about the features of aPF4/H Abs in the pathogenesis of HIT. Exploring the characteristics of HIT antibodies bears a potential to better understand general mechanisms of antibodymediated autoimmunity HIT. However, there is a difficulty in subtracting the pathogenic HIT antibody directly from human sera because both pathogenic and non-pathogenic antibodies bind to the PF4/H antigen.

Newman et al. reported that aPF4/P Abs can be purified by PF4-agarose beads [3]. Later in 2000, Amiral et al. described that affinity purification of aPF4/P Abs resulted in a mixture of IgA, IgM, and IgG [52]. In this mixture, only a subset of IgG antibodies activates platelets [49]. Contamination of IgA, IgM, and IgG antibodies will increase the difficulty in characterizing aPF4/P Abs. To overcome this limitation, two-step affinity chromatography has currently established to separate aPF4/H Abs from HIT patients sera. By this method, aPF4/P Abs from sera of patients were successfully isolated for three antibody groups. The purified Abs showed similar characteristics as the original serum in EIA and HIPA. Titrating the antibodies in ELISA, all antibody groups show an increase of OD with increasing antibody concentration (**Figure 5A**). OD values are highest for group-3, followed by group-2 and then group-1 Abs. In the HIPA test, group-1 Abs did not cause platelet aggregation up to a concentration of 89.7 μg/mL; group-2 Abs induced platelet aggregation from concentrations ≥43.5 μg/mL, but only in the presence of heparin; while group-3 Abs induced platelet aggregation from concentrations ≥5.2 μg/mL independently of heparin (**Figure 5B**). This is consistent with previous findings that chondroitin sulfate plays an important role in platelet activation by PF4/P Abs, even in the absence of heparin [53, 54].

**Figure 5.** Dose-dependent binding of aPF4/P Abs to PF4/H complexes in EIA and HIPA. (A) EIA shows the lowest OD of control IgG (black) as the background reaction, follow by group-1 (dark cyan), higher for group-2 (blue) and highest for group-3 (red) Abs. (B) HIPA tests show a dependence of platelet aggregation on antibody concentration: Group-1 Abs do not activate platelets, neither in the absence (−), nor in the presence (+) of reviparin up to a concentration of 89.7 μg/mL (dark cyan); group-2 Abs (blue) induced platelet activation (red part) at concentrations ≥44 μg/mL but only in the presence of reviparin; group-3 Abs (red) activated platelets at much lower concentrations (≥5 μg/mL) either in the presence or absence of reviparin. n = 5 sera per group. Adapted from [55].

#### *3.1.2. Binding strength of human-derived HIT antibodies*

The binding strength between the antibody and PF4/H complexes is determined by AFS. A single aPF4/H Abs is immobilized on the cantilever and then approach to the PF4/H complexes coated on a solid phase for interacting and measuring of their binding strength. Weakest binding forces were measured for monoclonal antibody KKO mimicking human HIT antibodies (43.6 ± 8.8 pN, gray) and group-1 Abs (44.0 ± 8.1 pN, green), higher for group-2 Abs (60.6 ± 15.4 pN, blue) and highest for group-3 Abs (72.4 ± 26.2 pN, red). Statistics showed no significant difference between KKO and group-1 Abs (p = 0.877), significant difference between group-1 and group-2 Abs (p < 0.001), or between group-2 and group-3 Abs (p = 0.006)) (**Figure 6**) [55].

even exceeded 100 pN. The low variability in binding forces of KKO and group-1 Abs has been attributed to the fact that they contain homogeneous antibodies, whereas the patient's sera such as group-2 and group-3 Abs contained polyclonal mixtures of aPF4/P Abs differently reactive. Among these human-derived Abs, it has been proved that the group-2 contains also antibodies reacting like group-1 Abs, while group-3 is highly complicated as it composes of not only antibodies reacting like group-1 and group-2 Abs but also some additional super strong reactive antibodies. The aPF4/H Abs show different reactivity patterns under various

**Figure 6.** Binding characteristics of aPF4/H Abs. Each dot shows the mean and standard error of the rupture force for each respective antibody from five sera per group. (A) KKO and (B) group-1 Abs bind to PF4/H complexes with a binding strength mostly ≤60 pN (black dotted line), while (C) group-2 and (C) group-3 Abs consist of Abs with different binding forces. (D) a subset of group-3 Abs binds to PF4/H complexes with rupture forces higher than 100 pN (red-

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The autoimmune group-3 Abs activate platelets in the absence of polyanions because they can self-cluster PF4 to form PF4/group-3 antibody complexes without the need of heparin [55]. This characteristic of autoimmune group-3 Abs has been proved by various methodologies: First, the autoimmune group-3 Abs could be purified from the patient's sera using a PF4 column (instead of the PF4/H column). Hardly any PF4/P Abs were obtained from control and group-1 sera; group-2 sera showed a minimally increased IgG yield. When these antibodies are concentrated to 50 μg/ml, only antibodies purified from group-3 sera activated platelets in the HIPA. The results indicate that group-3 sera contain antibodies with PF4 specificity,

pH and ionic strength conditions [56].

dotted line). Adapted from [55].

*3.1.3. Autoimmune antibodies cluster PF4*

which activate platelets.

Group-3 Abs bound to PF4/H complexes with much higher binding energy (ΔH = −2.87 ± 2.06 × 10<sup>8</sup> cal/mol) than group-2 Abs (ΔH = −2.90 ± 0.4 × 10<sup>4</sup> cal/mol), and their dissociation constant (KD) (~5.3 nM) was about two orders of magnitude lower than that of group-2 Abs (~1.7 × 102 nM). The binding strength of PF4 to heparin ~150 pN [35] is higher than that between group-3 Abs and PF4/H complexes (mostly lower than 150 pN) [55]. Besides that, the group-3 Abs have a highest binding affinity (koff = 0.12 s−1) as compared with group-1 Abs y (koff = 15.6 s−1), group-2 Abs (koff = 2.0 s−1), or KKO (koff = 2.2 s−1). The lowest thermal offrate specify that multiplexes induced by PF4/H complexes with group-3 Abs are more stable than those formed with other antibody groups. Furthermore, KKO and group-1 Abs contain antibodies with similar characteristics, and therefore, they interacted rather uniformly with PF4/H complexes. This has been clarified by obtaining the relatively small differences among the rupture forces (<60 pN, **Figure 6A**-**B**) measured from different cantilevers. However, group-2 Abs contain different types of antibodies as observed by a large variation of all binding forces (~40% exceeded 60 pN). For group-3 Abs, the variation of binding force is even higher than that of group-2 Abs as shown by ~44% of all binding forces ≥60 pN and ~15%

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**Figure 6.** Binding characteristics of aPF4/H Abs. Each dot shows the mean and standard error of the rupture force for each respective antibody from five sera per group. (A) KKO and (B) group-1 Abs bind to PF4/H complexes with a binding strength mostly ≤60 pN (black dotted line), while (C) group-2 and (C) group-3 Abs consist of Abs with different binding forces. (D) a subset of group-3 Abs binds to PF4/H complexes with rupture forces higher than 100 pN (reddotted line). Adapted from [55].

even exceeded 100 pN. The low variability in binding forces of KKO and group-1 Abs has been attributed to the fact that they contain homogeneous antibodies, whereas the patient's sera such as group-2 and group-3 Abs contained polyclonal mixtures of aPF4/P Abs differently reactive. Among these human-derived Abs, it has been proved that the group-2 contains also antibodies reacting like group-1 Abs, while group-3 is highly complicated as it composes of not only antibodies reacting like group-1 and group-2 Abs but also some additional super strong reactive antibodies. The aPF4/H Abs show different reactivity patterns under various pH and ionic strength conditions [56].

#### *3.1.3. Autoimmune antibodies cluster PF4*

*3.1.2. Binding strength of human-derived HIT antibodies*

40 Thrombocytopenia

presence or absence of reviparin. n = 5 sera per group. Adapted from [55].

The binding strength between the antibody and PF4/H complexes is determined by AFS. A single aPF4/H Abs is immobilized on the cantilever and then approach to the PF4/H complexes coated on a solid phase for interacting and measuring of their binding strength. Weakest binding forces were measured for monoclonal antibody KKO mimicking human HIT antibodies (43.6 ± 8.8 pN, gray) and group-1 Abs (44.0 ± 8.1 pN, green), higher for group-2 Abs (60.6 ± 15.4 pN, blue) and highest for group-3 Abs (72.4 ± 26.2 pN, red). Statistics showed no significant difference between KKO and group-1 Abs (p = 0.877), significant difference between group-1 and group-2 Abs (p < 0.001), or between group-2 and group-3 Abs (p = 0.006)) (**Figure 6**) [55]. Group-3 Abs bound to PF4/H complexes with much higher binding energy (ΔH = −2.87 ± 2.06 × 10<sup>8</sup> cal/mol) than group-2 Abs (ΔH = −2.90 ± 0.4 × 10<sup>4</sup> cal/mol), and their dissociation constant (KD) (~5.3 nM) was about two orders of magnitude lower than that of group-2 Abs (~1.7 × 102 nM). The binding strength of PF4 to heparin ~150 pN [35] is higher than that between group-3 Abs and PF4/H complexes (mostly lower than 150 pN) [55]. Besides that, the group-3 Abs have a highest binding affinity (koff = 0.12 s−1) as compared with group-1 Abs y (koff = 15.6 s−1), group-2 Abs (koff = 2.0 s−1), or KKO (koff = 2.2 s−1). The lowest thermal offrate specify that multiplexes induced by PF4/H complexes with group-3 Abs are more stable than those formed with other antibody groups. Furthermore, KKO and group-1 Abs contain antibodies with similar characteristics, and therefore, they interacted rather uniformly with PF4/H complexes. This has been clarified by obtaining the relatively small differences among the rupture forces (<60 pN, **Figure 6A**-**B**) measured from different cantilevers. However, group-2 Abs contain different types of antibodies as observed by a large variation of all binding forces (~40% exceeded 60 pN). For group-3 Abs, the variation of binding force is even higher than that of group-2 Abs as shown by ~44% of all binding forces ≥60 pN and ~15%

**Figure 5.** Dose-dependent binding of aPF4/P Abs to PF4/H complexes in EIA and HIPA. (A) EIA shows the lowest OD of control IgG (black) as the background reaction, follow by group-1 (dark cyan), higher for group-2 (blue) and highest for group-3 (red) Abs. (B) HIPA tests show a dependence of platelet aggregation on antibody concentration: Group-1 Abs do not activate platelets, neither in the absence (−), nor in the presence (+) of reviparin up to a concentration of 89.7 μg/mL (dark cyan); group-2 Abs (blue) induced platelet activation (red part) at concentrations ≥44 μg/mL but only in the presence of reviparin; group-3 Abs (red) activated platelets at much lower concentrations (≥5 μg/mL) either in the

> The autoimmune group-3 Abs activate platelets in the absence of polyanions because they can self-cluster PF4 to form PF4/group-3 antibody complexes without the need of heparin [55]. This characteristic of autoimmune group-3 Abs has been proved by various methodologies:

> First, the autoimmune group-3 Abs could be purified from the patient's sera using a PF4 column (instead of the PF4/H column). Hardly any PF4/P Abs were obtained from control and group-1 sera; group-2 sera showed a minimally increased IgG yield. When these antibodies are concentrated to 50 μg/ml, only antibodies purified from group-3 sera activated platelets in the HIPA. The results indicate that group-3 sera contain antibodies with PF4 specificity, which activate platelets.

Next, only autoantibodies (group-3) show strong interaction with PF4 alone by ITC. When the antibodies were tested at the same concentration of 62.5 nM, KKO and group-2 Abs did not interact with PF4, while group-3 Abs interacted strongly. As the interaction between group-3 Abs and PF4 alone showed two binding sites (stoichiometry n = CABS/CPF4 = 0.53 ± 0.003), these Abs can cluster two PF4 molecules. Increasing antibody concentration did not improve binding of KKO to PF4 whereas group-2 Abs weakly interacted with PF4. However, the binding energy released by group-2 Abs is only 0.1% compared to that of group-3 Abs.

Consistently, PF4 or PF4/H EIA show that group-3 Abs bound quite strong to PF4 while other antibodies did not even though all Abs bound much stronger to PF4/H complexes than to PF4 alone. By AFS, group-1 and group-2 Abs showed much less binding events to PF4 than to PF4/H complexes, while the super-reactive group-3 Abs showed similar bindings. In addition, the interaction forces of group-3 Abs purified via a PF4-column with PF4/H complexes showed the highest range of binding forces (~100 pN). These results again indicate that group-3 Abs bind strongly to PF4 alone independently from heparin, while bindings of group-1 and group-2 Abs are heparin-dependent.

By dynamic light scattering (DLS), group-3 Abs formed the largest complexes with PF4 as compared to other antibody groups with even larger size than PF4/H complexes further indicate that group-3 Abs can cluster PF4.

The binding energy generated by the interaction of group-3 Abs with PF4 in the ITC experiments (ΔH = −3.5 ± 0.86 × 10<sup>7</sup> cal/mol) is much higher than the energy released when a 16-mer heparin interacts with PF4 (ΔH = −7.26 ± 1.36 × 10<sup>3</sup> cal/mol) [6]. As 16-mer heparin can force two PF4 molecules together, based on their high energy release, group-3 Abs most probably also can force two PF4 tetramers together. In addition, the negative entropy of the reaction (ΔS = −11.7 ± 2.8 × 10<sup>4</sup> cal/mol. K) is attributed to PF4 conformational change when forming complexes with the group-3 Abs. By DLS, the size complexes formed by PF4 and group-3 Abs increases significantly when the group-2 Abs are added, indicating that group-3 Abs, induce a conformational change in PF4 and the resulting PF4/group-3 antibody complexes allow binding of group-2 Abs in the same way as polyanions do.

and has been used to understand the binding characteristics of an antibody recognizing PF4/P complexes and activating platelets [62, 63]. Binding of a non-HIT antibody RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation/aggregation in vitro, and thrombus progression in vivo. The probability and the interaction force of KKO binding to PF4 are much greater than those of RTO, while KKO/ PF4 dissociation rate was approximately 10-fold slower than RTO/PF4 [62, 63], indicating that KKO binds stronger than RTO and KKO/PF4 complexes are more stable than RTO/PF4.

**Figure 7.** Group-3 Abs cluster PF4 and enhance platelet activation. (A) PF4 form large complexes with heparin and the resulting PF4/H complexes allow (B) group-2 Abs bind and (C) induce platelet aggregation/activation. (D) a subset of group-3 Abs cluster PF4 forming PF4/Group-3 antibody complexes which also (E) allow binding of group-2 Abs and (F) enhance platelet aggregation/activation evidenced by tighter and denser aggregates compared to (C). Adapted from [55].

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KKO interacts with PF4/H complexes coated platelets with ~4-fold higher forces than with PF4/H complexes coated on a solid phase, while RTO shows only a minor change [64]. The different binding forces strongly indicate that PF4 and PF4/H complexes either expose different epitopes or allow better access of platelet-activating Abs to their epitope when PF4 bound to the platelet surface compared to the presentation of PF4/H complexes on a solid phase. Most probably, PF4/H complexes exhibited the antigenic site differently depending on the bound substrates [53]. The findings provide an explanation for the surprising observation that KKO interact relatively weak when PF4/H complexes are immobilized on a solid phase [55], while it strongly activates platelets in functional assays. It is unresolved, which additional binding partners on the platelet surface interfere with the conformational change or different presentations of PF4/H complexes. Nevertheless, chondroitin sulfate [53] and polyphosphates

[65] are potential candidates, as they interact with PF4.

Altogether, PF4 form large complexes with heparin and allow group-2 Abs bind and induce platelet aggregation/activation (**Figure 7A**-**C**). Importantly, a subset of group-3 Abs cluster PF4 and the resulting PF4/Group-3 antibody complexes also allow binding of group-2 Abs and enhance platelet aggregation/activation even stronger than heparins do as shown by tighter and denser aggregates (**Figure 7D**-**F**).

### **3.2. HIT-like antibodies**

Many studies in HIT have been performed with human aPF4/P Abs isolated from patient plasma because only one monoclonal antibody (KKO) mimicking human HIT antibodies did exist until recently [57]. KKO activates platelets [58] and monocytes [59] *in vitro* and *in vivo* by cross-linking FcγRIIa. KKO has been used to unravel the pathogenesis of HIT and is the basis for a recently FDA approved plasma-based antigen assay (HIT-HemosIL) for detection of PF4/P antibodies [60, 61]. KKO mimics the biological activity of human aPF4/P Abs [62]

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Next, only autoantibodies (group-3) show strong interaction with PF4 alone by ITC. When the antibodies were tested at the same concentration of 62.5 nM, KKO and group-2 Abs did not interact with PF4, while group-3 Abs interacted strongly. As the interaction between group-3 Abs and PF4 alone showed two binding sites (stoichiometry n = CABS/CPF4 = 0.53 ± 0.003), these Abs can cluster two PF4 molecules. Increasing antibody concentration did not improve binding of KKO to PF4 whereas group-2 Abs weakly interacted with PF4. However, the binding

Consistently, PF4 or PF4/H EIA show that group-3 Abs bound quite strong to PF4 while other antibodies did not even though all Abs bound much stronger to PF4/H complexes than to PF4 alone. By AFS, group-1 and group-2 Abs showed much less binding events to PF4 than to PF4/H complexes, while the super-reactive group-3 Abs showed similar bindings. In addition, the interaction forces of group-3 Abs purified via a PF4-column with PF4/H complexes showed the highest range of binding forces (~100 pN). These results again indicate that group-3 Abs bind strongly to PF4 alone independently from heparin, while bindings of

By dynamic light scattering (DLS), group-3 Abs formed the largest complexes with PF4 as compared to other antibody groups with even larger size than PF4/H complexes further indi-

The binding energy generated by the interaction of group-3 Abs with PF4 in the ITC experi-

heparin interacts with PF4 (ΔH = −7.26 ± 1.36 × 10<sup>3</sup> cal/mol) [6]. As 16-mer heparin can force two PF4 molecules together, based on their high energy release, group-3 Abs most probably also can force two PF4 tetramers together. In addition, the negative entropy of the reaction (ΔS = −11.7 ± 2.8 × 10<sup>4</sup> cal/mol. K) is attributed to PF4 conformational change when forming complexes with the group-3 Abs. By DLS, the size complexes formed by PF4 and group-3 Abs increases significantly when the group-2 Abs are added, indicating that group-3 Abs, induce a conformational change in PF4 and the resulting PF4/group-3 antibody complexes allow bind-

Altogether, PF4 form large complexes with heparin and allow group-2 Abs bind and induce platelet aggregation/activation (**Figure 7A**-**C**). Importantly, a subset of group-3 Abs cluster PF4 and the resulting PF4/Group-3 antibody complexes also allow binding of group-2 Abs and enhance platelet aggregation/activation even stronger than heparins do as shown by

Many studies in HIT have been performed with human aPF4/P Abs isolated from patient plasma because only one monoclonal antibody (KKO) mimicking human HIT antibodies did exist until recently [57]. KKO activates platelets [58] and monocytes [59] *in vitro* and *in vivo* by cross-linking FcγRIIa. KKO has been used to unravel the pathogenesis of HIT and is the basis for a recently FDA approved plasma-based antigen assay (HIT-HemosIL) for detection of PF4/P antibodies [60, 61]. KKO mimics the biological activity of human aPF4/P Abs [62]

cal/mol) is much higher than the energy released when a 16-mer

energy released by group-2 Abs is only 0.1% compared to that of group-3 Abs.

group-1 and group-2 Abs are heparin-dependent.

ing of group-2 Abs in the same way as polyanions do.

tighter and denser aggregates (**Figure 7D**-**F**).

**3.2. HIT-like antibodies**

cate that group-3 Abs can cluster PF4.

ments (ΔH = −3.5 ± 0.86 × 10<sup>7</sup>

42 Thrombocytopenia

**Figure 7.** Group-3 Abs cluster PF4 and enhance platelet activation. (A) PF4 form large complexes with heparin and the resulting PF4/H complexes allow (B) group-2 Abs bind and (C) induce platelet aggregation/activation. (D) a subset of group-3 Abs cluster PF4 forming PF4/Group-3 antibody complexes which also (E) allow binding of group-2 Abs and (F) enhance platelet aggregation/activation evidenced by tighter and denser aggregates compared to (C). Adapted from [55].

and has been used to understand the binding characteristics of an antibody recognizing PF4/P complexes and activating platelets [62, 63]. Binding of a non-HIT antibody RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation/aggregation in vitro, and thrombus progression in vivo. The probability and the interaction force of KKO binding to PF4 are much greater than those of RTO, while KKO/ PF4 dissociation rate was approximately 10-fold slower than RTO/PF4 [62, 63], indicating that KKO binds stronger than RTO and KKO/PF4 complexes are more stable than RTO/PF4.

KKO interacts with PF4/H complexes coated platelets with ~4-fold higher forces than with PF4/H complexes coated on a solid phase, while RTO shows only a minor change [64]. The different binding forces strongly indicate that PF4 and PF4/H complexes either expose different epitopes or allow better access of platelet-activating Abs to their epitope when PF4 bound to the platelet surface compared to the presentation of PF4/H complexes on a solid phase. Most probably, PF4/H complexes exhibited the antigenic site differently depending on the bound substrates [53]. The findings provide an explanation for the surprising observation that KKO interact relatively weak when PF4/H complexes are immobilized on a solid phase [55], while it strongly activates platelets in functional assays. It is unresolved, which additional binding partners on the platelet surface interfere with the conformational change or different presentations of PF4/H complexes. Nevertheless, chondroitin sulfate [53] and polyphosphates [65] are potential candidates, as they interact with PF4.

However, KKO is a mouse IgG2b antibody (an absent subclass in humans) [66], while the platelet-activating aPF4/P Abs present in HIT plasma samples are predominantly IgG1. KKO behaves differently from human aPF4/P Abs, i.e. it binds only weakly to PF4/H complexes coated on a solid phase [64]. Recently, a chimeric monoclonal aPF4/H Abs with a human Fc fragment (5B9) has been developed [67]. The 5B9 antibody has been demonstrated to fully mimic the cellular effects of human HIT Abs [10, 68].

binding of otherwise aPF4/P Abs. The resulting immunocomplexes induce massive platelet activation in the absence of heparin. The source and length of heparins play an important role in inducing thrombocytopenia. Improvement of heparin quality together with discovering new non-heparin drugs should be highly desirable. Patients who are suspected of HIT need to be immediately stopped heparin exposure and switched to an alternative anticoagulant. Regarding patients with antibody-induced thrombocytopenia, the level of complication is much higher than the general heparin-induced thrombocytopenia. To date, these humanderived antibodies are hardly controlled, and therefore, efforts in the field would be appreciated. Clinical tests for detecting HIT antibodies as well as autoimmune HIT antibodies must

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This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Germany) (NG

Institute of Immunology and Transfusion Medicine, University Medicine Greifswald,

[1] Greinacher A. Heparin-induced thrombocytopenia. The New England Journal of Medi-

[2] Warkentin TE, Greinacher A. Heparin-Induced Thrombocytopenia. 4th ed. CRC Press;

[3] Blank M, Shoenfeld Y, Tavor S, Praprotnik S, Boffa MC, Weksler B, Walenga MJ, Amiral J, Eldor A. Anti-platelet factor 4/heparin antibodies from patients with heparin-induced thrombocytopenia provoke direct activation of microvascular endothelial cells. Interna-

be improved to achieve an appropriate identification of clinical HIT patients.

**Acknowledgements**

**Conflict of interest**

**Author details**

Thi-Huong Nguyen

Greifswald, Germany

cine. 2015;**373**(19):1883-1884

tional Immunology. 2002;**14**(2):121-129

**References**

2013

The authors declare no competing financial interests.

Address all correspondence to: nguyent@uni-greifswald.de

133/1-1).
