**1. Introduction**

Heparin-induced thrombocytopenia (HIT) as a severe adverse drug effect occurs when patients receive heparin anticoagulant to prevent and treat thromboembolic diseases. Depending on the length of heparin, HIT occurs in ≤5% of patients receiving high molecular weight unfractionated heparin, whereas ≤1% of patients receiving low molecular weight heparin. In HIT, the immune system considers the platelet factor 4 (PF4), which is altered in its conformation after binding to heparin (H), to be "foreign" and the formation of anti-PF4/H antibodies (aPF4/P Abs) occurs. Upon binding to the PF4/H complex, these antibodies activate circulating platelets and other cells. Typically, 5–14 days after heparin exposure, platelet count reduces to <15–20 × 10<sup>9</sup> cells/L (or a > 50% decrease in platelet count). HIT can result

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in thromboses such as deep vein thrombosis (DVT), pulmonary embolism (PE), occlusion of a limb artery, acute myocardial infarct, stroke, and a systemic reaction or skin necrosis. Importantly, there is also a subset of anti-PF4/Heparin antibodies (aPF4/H Abs) which, in the absence of heparin, can lead to symptomatic thrombocytopenia and excessive vascular thrombosis. The extreme sequela of the aPF4/H Abs is autoimmune HIT, in which individuals develop multiple vessel occlusions without drug exposure.

of an aPF4/P Ab to a PF4/H complex contains two platelet binding sites, that is, one is on the PF4/H complexes, and another one is on the Fc part of the IgG which binds to FcγRIIa receptors [9, 10] on platelet membranes (**Figure 1C**). Cross-linking of the platelet Fc receptor results in platelet activation that releases more PF4s and facilitates formation additional ultra large immune complexes. These complexes rapidly recruit other platelets into the prothrombotic process (**Figure 1D**). Activation of platelets leads to the loss of platelets, massive platelet activation and even triggers clotting cascade that results in thrombin generation and increases the risk for vessel occlusions such as venous thrombosis, myocardial infarction or stroke [7, 11, 12]. The binding strength of a blood thrombus has major biological importance. A recent study could determine directly the binding strength between two platelets at single platelet level [13]. The binding force increases proportionally to the degree of platelet activation but reduces with blockade of specific platelet receptors. The method provides major perspectives for testing and improving the biocompatibility of new materials, quantifying the effect of drugs on platelet function, and assessing the mechanical characteristics of acquired/inherited platelet defects. Heparins are the glycosaminoglycans (GAGs) containing glucosamine residues with a high degree of sulfation that dictates their biological activities [6, 14, 15]. GAGs play an important role in the sequestration of plasmodium falciparum-infected red blood cells in the microvascular endothelium of different tissues [16, 17]. Their pharmacologic activity is mediated by a chemically unique pentasaccharide sequence present in about 30% of all heparin molecules. Heparin behaves like simple entropic spring forces, which is produced by sugar rings of heparin flipping to more energetic and more extended conformations [18, 19]. Both low and high molecular weight heparins are available. The source of high molecular weight unfractionated heparin (UFH) influences the risk of HIT, i.e. bovine UFH is more likely to cause HIT than porcine UFH [20–22]. Besides UFH, the low molecular weight heparins (LMWH) produced from UFH by chemical fractionation, are widely used in clinical practice [23–27]. Due to their shorter chain length, LMWHs show less strong interaction with PF4. UFH and PF4 form ultra large complexes (ULCs) when both are present approximately at an optimal 1:1 ratio. Comparing with UFH, LMWHs form smaller complexes with PF4. ULCs showed a greater capacity to promote platelet activation than small complexes [28]. These differences in complex formation between UFH and LMWHs translate into their risk for inducing HIT in patients. LMWHs induce HIT about 10 times less frequent than UFH, but HIT still randomly occurs during treatment with LMWHs [29–32].

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**2.1. Boundary between antigenic and non-antigenic heparin**

PF4/heparin (or polyanions) complexes can become antigenic or not depend on heparin (or polyanion) characteristics. To expose neoepitopes on PF4s relevant for HIT, at least three bonds between the polyanion and PF4 in the PF4/polyanion complex should be formed [33]. These neoepitopes on the PF4/polyanion complexes then allow binding of the aPF4/H Abs. The binding strength of the single sulfate groups on the polyanion with the PF4 does not differ among polyanions with a different degree of sulfation [33]. The quantity and resulting density of sulfate groups on the polyanion chain determine their molecular effects on PF4 [33]. In particular, the polyanions which bind to PF4 tetramer with less than three sulfate bonds are unable to expose the neoepitope [6, 34]. The results suggest an existence of a boundary between antigenic (risk for HIT) and non-antigenic heparins (non-risk for HIT). This boundary has been determined by

### **2. Heparin-induced thrombocytopenia**

Heparin-induced thrombocytopenia (HIT) is a distinct clinicopathologic syndrome caused by platelet-activating antibodies that bridge between complexes of platelet factor 4-Heparin (PF4/H) and platelets [1, 2] or endothelial cells [3]. Human platelets are anuclear cell fragments with discoidal shapes of 1–2 μm, originating from the cytoplasm of bone marrow megakaryocytes [4]. Platelets store PF4 (a positively charged tetramer belonging to CXC chemokine family) in their alpha granules. Non-activated platelets release some PF4s (**Figure 1A**) [5]. When patients take anticoagulant polyanions like heparin, some of these heparins bind to PF4s forming ultra large PF4/H complexes (**Figure 1B**). Binding of heparin to PF4 induces a conformational change in PF4s [6–8] which results in an expression of new epitopes. Some patients develop antibodies against these neoepitopes (**Figure 1B**). These human-derived antibodies are defined as anti-PF4/H antibodies (aPF4/P Abs). Each resulting multimolecular complex

**Figure 1.** Cartoon illustrates the formation of heparin-induced thrombocytopenia (HIT). (A) Non-activated platelets secrete several PF4s. (B) with heparin exposure, PF4s form ultra large complexes with long heparins that induce conformational changes in PF4s. Some patients develop aPF4/H Abs against PF4 neoepitopes. (C) Human-derived aPF4/H Abs bound PF4/H complexes can adhere to platelet membrane. (D) Fc parts of the antibodies link fcγRIIa receptors on platelet membranes that leads to platelet aggregation/activation. Adapted from [5].

of an aPF4/P Ab to a PF4/H complex contains two platelet binding sites, that is, one is on the PF4/H complexes, and another one is on the Fc part of the IgG which binds to FcγRIIa receptors [9, 10] on platelet membranes (**Figure 1C**). Cross-linking of the platelet Fc receptor results in platelet activation that releases more PF4s and facilitates formation additional ultra large immune complexes. These complexes rapidly recruit other platelets into the prothrombotic process (**Figure 1D**). Activation of platelets leads to the loss of platelets, massive platelet activation and even triggers clotting cascade that results in thrombin generation and increases the risk for vessel occlusions such as venous thrombosis, myocardial infarction or stroke [7, 11, 12]. The binding strength of a blood thrombus has major biological importance. A recent study could determine directly the binding strength between two platelets at single platelet level [13]. The binding force increases proportionally to the degree of platelet activation but reduces with blockade of specific platelet receptors. The method provides major perspectives for testing and improving the biocompatibility of new materials, quantifying the effect of drugs on platelet function, and assessing the mechanical characteristics of acquired/inherited platelet defects.

in thromboses such as deep vein thrombosis (DVT), pulmonary embolism (PE), occlusion of a limb artery, acute myocardial infarct, stroke, and a systemic reaction or skin necrosis. Importantly, there is also a subset of anti-PF4/Heparin antibodies (aPF4/H Abs) which, in the absence of heparin, can lead to symptomatic thrombocytopenia and excessive vascular thrombosis. The extreme sequela of the aPF4/H Abs is autoimmune HIT, in which individuals

Heparin-induced thrombocytopenia (HIT) is a distinct clinicopathologic syndrome caused by platelet-activating antibodies that bridge between complexes of platelet factor 4-Heparin (PF4/H) and platelets [1, 2] or endothelial cells [3]. Human platelets are anuclear cell fragments with discoidal shapes of 1–2 μm, originating from the cytoplasm of bone marrow megakaryocytes [4]. Platelets store PF4 (a positively charged tetramer belonging to CXC chemokine family) in their alpha granules. Non-activated platelets release some PF4s (**Figure 1A**) [5]. When patients take anticoagulant polyanions like heparin, some of these heparins bind to PF4s forming ultra large PF4/H complexes (**Figure 1B**). Binding of heparin to PF4 induces a conformational change in PF4s [6–8] which results in an expression of new epitopes. Some patients develop antibodies against these neoepitopes (**Figure 1B**). These human-derived antibodies are defined as anti-PF4/H antibodies (aPF4/P Abs). Each resulting multimolecular complex

**Figure 1.** Cartoon illustrates the formation of heparin-induced thrombocytopenia (HIT). (A) Non-activated platelets secrete several PF4s. (B) with heparin exposure, PF4s form ultra large complexes with long heparins that induce conformational changes in PF4s. Some patients develop aPF4/H Abs against PF4 neoepitopes. (C) Human-derived aPF4/H Abs bound PF4/H complexes can adhere to platelet membrane. (D) Fc parts of the antibodies link fcγRIIa

receptors on platelet membranes that leads to platelet aggregation/activation. Adapted from [5].

develop multiple vessel occlusions without drug exposure.

**2. Heparin-induced thrombocytopenia**

34 Thrombocytopenia

Heparins are the glycosaminoglycans (GAGs) containing glucosamine residues with a high degree of sulfation that dictates their biological activities [6, 14, 15]. GAGs play an important role in the sequestration of plasmodium falciparum-infected red blood cells in the microvascular endothelium of different tissues [16, 17]. Their pharmacologic activity is mediated by a chemically unique pentasaccharide sequence present in about 30% of all heparin molecules. Heparin behaves like simple entropic spring forces, which is produced by sugar rings of heparin flipping to more energetic and more extended conformations [18, 19]. Both low and high molecular weight heparins are available. The source of high molecular weight unfractionated heparin (UFH) influences the risk of HIT, i.e. bovine UFH is more likely to cause HIT than porcine UFH [20–22]. Besides UFH, the low molecular weight heparins (LMWH) produced from UFH by chemical fractionation, are widely used in clinical practice [23–27]. Due to their shorter chain length, LMWHs show less strong interaction with PF4. UFH and PF4 form ultra large complexes (ULCs) when both are present approximately at an optimal 1:1 ratio. Comparing with UFH, LMWHs form smaller complexes with PF4. ULCs showed a greater capacity to promote platelet activation than small complexes [28]. These differences in complex formation between UFH and LMWHs translate into their risk for inducing HIT in patients. LMWHs induce HIT about 10 times less frequent than UFH, but HIT still randomly occurs during treatment with LMWHs [29–32].

#### **2.1. Boundary between antigenic and non-antigenic heparin**

PF4/heparin (or polyanions) complexes can become antigenic or not depend on heparin (or polyanion) characteristics. To expose neoepitopes on PF4s relevant for HIT, at least three bonds between the polyanion and PF4 in the PF4/polyanion complex should be formed [33]. These neoepitopes on the PF4/polyanion complexes then allow binding of the aPF4/H Abs. The binding strength of the single sulfate groups on the polyanion with the PF4 does not differ among polyanions with a different degree of sulfation [33]. The quantity and resulting density of sulfate groups on the polyanion chain determine their molecular effects on PF4 [33]. In particular, the polyanions which bind to PF4 tetramer with less than three sulfate bonds are unable to expose the neoepitope [6, 34]. The results suggest an existence of a boundary between antigenic (risk for HIT) and non-antigenic heparins (non-risk for HIT). This boundary has been determined by applying multiple techniques such as atomic force microscopy-based atomic force microscopy (AFS) [35], isothermal titration calorimetry (ITC) [6], or circular dichroism (CD) spectroscopy in combination with enzyme-linked immunosorbent assay (ELISA) [7] (**Figure 2**).

AFS shows that both numbers of specific rupture events and magnitude of rupture forces rise with an increase of heparin length, suggesting that long heparins form with PF4 more bonds than short ones [35]. A larger variation of the rupture forces for long heparins ≥8-mer compared with short heparins ≤6-mer was observed (**Figure 3A**). The enthalpy obtained by ITC rises with the increase of heparin length and reaches maximal values at ~11-mer (**Figure 3B**) [36]. Combining the results obtained by AFS and ITC, the boundary between non-antigenic and antigenic heparin is determined between 8- to 11-mer. This boundary is further clarified by CD spectroscopy which is sensitive to the secondary structure and folding properties of proteins [37]. For PF4/H interactions, the change in β-sheet content was found to be ≤30% for short heparin and >30% for long heparins (**Figure 3C**). By ELISA, optical density (OD) was ≤0.5 for short heparin, while OD was >0.5 for longer heparins (>8-mer) (**Figure 3C**). The OD of 0.5 is the threshold to determine whether a heparin used in the ELISA was able to support binding of aPF4/H Abs. The combination of β-sheet content and OD values show clearly a dissimilar behavior between short and long heparins (**Figure 3C**).

Linking together all the results from AFS, ITC, CD spectroscopy and ELISA, the boundary between antigenic and non-antigenic heparin has been proved between 8- and 11-mer. These findings are particularly important to understand PF4-Heparin binding processes and to develop new heparin-derived drugs with reduced risk for adverse immune reactions.

**Figure 3.** Model describing different binding pathways between short and long heparins when interacting with PF4 tetramers. (A) Depending on heparin length, short heparin can bind to one PF4 tetramer, (B) whereas long heparin bridges two PF4s and forces them closer to each other at a distance l < L, merging two hydrophobic surfaces of PF4s

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Thermodynamic and kinetic parameters of the ligand-receptor interactions can be obtained by applying the Bell-Evans [38] or the Friddle [39, 40] models. The models show that the faster the molecule is pulled, the higher the rupture force will be measured. For simple ligandreceptor interaction in which multiple interactions are not involved, the rupture force (F) increases proportionally to the logarithmic loading rate. Even though there is some variation in the parameters obtained by these two models, Bell-Evans model is still a powerful tool to determine the kinetics of ligand-receptor interactions [41, 42]. For the PF4/H system, the PF4 tetramer is considered as one antigen or the interaction between heparin and PF4 is formed by a single bond, and therefore, applicable to the Bell-Evans model [35]. Short heparins show higher koff values than long heparins, indicating that PF4/long heparin complexes are more stable than PF4/short heparin complexes (**Table 1**). With binding affinity (KA) measured by ITC [6], the thermal on-rate (kon = koff. KA) of PF4/H complexes is calculated. The short hepa-

PF4-Heparins interaction is more complex than general ligand-receptor interactions which are attributed to the electrostatic attraction. Based on special features in force-distance curves and the magnitude of PF4/H binding forces, it has been proved that long heparin bound PF4s creating additional PF4-PF4 bonds [35]. Long heparins form two types of bonds with PFs, i.e.

Combination of these techniques allows better characterizing heparin boundary.

**2.2. Kinetic properties and binding model of PF4/H complexes**

(green shaded area). Adapted from [35].

rins bind to PF4s with ~10–20 times faster than long heparins [35].

**Parameter HO06 HO12 HO16** koff (s−1) 1.64 1.40 × 10−2 1.10 × 10−4 kon(M−1 s−1) 0.41 × 105 0.32 × 10<sup>4</sup> 0.55 × 10<sup>3</sup> ΔE (kBT) −0.49 4.27 9.12

**Table 1.** Thermodynamic and kinetic parameters of PF4/heparin interactions [35].

**Figure 2.** Determination of the boundary between antigenic and non-antigenic heparins. (A) Rupture force histograms fitted by Gaussian distributions show narrow widths (green arrows) for heparins ≤6-mer (HO05, HO06) and wider widths for longer heparins ≥8-mer (HO08, HO012, HO016). (B) ITC demonstrates lower enthalpy for short heparins (black dotted box) and higher enthalpy for long heparins (red-dotted box), while a saturation is found at ~11-mer. (C) Combination of CD spectroscopy and EIA shows that a boundary between short and long heparins is at ~30% ß-sheet contents and OD ~0.5. Overall, the boundary is determined between 8- and 11-mer. Adapted from [8, 35, 48].

**Figure 3.** Model describing different binding pathways between short and long heparins when interacting with PF4 tetramers. (A) Depending on heparin length, short heparin can bind to one PF4 tetramer, (B) whereas long heparin bridges two PF4s and forces them closer to each other at a distance l < L, merging two hydrophobic surfaces of PF4s (green shaded area). Adapted from [35].

Linking together all the results from AFS, ITC, CD spectroscopy and ELISA, the boundary between antigenic and non-antigenic heparin has been proved between 8- and 11-mer. These findings are particularly important to understand PF4-Heparin binding processes and to develop new heparin-derived drugs with reduced risk for adverse immune reactions. Combination of these techniques allows better characterizing heparin boundary.

#### **2.2. Kinetic properties and binding model of PF4/H complexes**

applying multiple techniques such as atomic force microscopy-based atomic force microscopy (AFS) [35], isothermal titration calorimetry (ITC) [6], or circular dichroism (CD) spectroscopy in

AFS shows that both numbers of specific rupture events and magnitude of rupture forces rise with an increase of heparin length, suggesting that long heparins form with PF4 more bonds than short ones [35]. A larger variation of the rupture forces for long heparins ≥8-mer compared with short heparins ≤6-mer was observed (**Figure 3A**). The enthalpy obtained by ITC rises with the increase of heparin length and reaches maximal values at ~11-mer (**Figure 3B**) [36]. Combining the results obtained by AFS and ITC, the boundary between non-antigenic and antigenic heparin is determined between 8- to 11-mer. This boundary is further clarified by CD spectroscopy which is sensitive to the secondary structure and folding properties of proteins [37]. For PF4/H interactions, the change in β-sheet content was found to be ≤30% for short heparin and >30% for long heparins (**Figure 3C**). By ELISA, optical density (OD) was ≤0.5 for short heparin, while OD was >0.5 for longer heparins (>8-mer) (**Figure 3C**). The OD of 0.5 is the threshold to determine whether a heparin used in the ELISA was able to support binding of aPF4/H Abs. The combination of β-sheet content and OD values show clearly a

**Figure 2.** Determination of the boundary between antigenic and non-antigenic heparins. (A) Rupture force histograms fitted by Gaussian distributions show narrow widths (green arrows) for heparins ≤6-mer (HO05, HO06) and wider widths for longer heparins ≥8-mer (HO08, HO012, HO016). (B) ITC demonstrates lower enthalpy for short heparins (black dotted box) and higher enthalpy for long heparins (red-dotted box), while a saturation is found at ~11-mer. (C) Combination of CD spectroscopy and EIA shows that a boundary between short and long heparins is at ~30% ß-sheet

contents and OD ~0.5. Overall, the boundary is determined between 8- and 11-mer. Adapted from [8, 35, 48].

combination with enzyme-linked immunosorbent assay (ELISA) [7] (**Figure 2**).

36 Thrombocytopenia

dissimilar behavior between short and long heparins (**Figure 3C**).

Thermodynamic and kinetic parameters of the ligand-receptor interactions can be obtained by applying the Bell-Evans [38] or the Friddle [39, 40] models. The models show that the faster the molecule is pulled, the higher the rupture force will be measured. For simple ligandreceptor interaction in which multiple interactions are not involved, the rupture force (F) increases proportionally to the logarithmic loading rate. Even though there is some variation in the parameters obtained by these two models, Bell-Evans model is still a powerful tool to determine the kinetics of ligand-receptor interactions [41, 42]. For the PF4/H system, the PF4 tetramer is considered as one antigen or the interaction between heparin and PF4 is formed by a single bond, and therefore, applicable to the Bell-Evans model [35]. Short heparins show higher koff values than long heparins, indicating that PF4/long heparin complexes are more stable than PF4/short heparin complexes (**Table 1**). With binding affinity (KA) measured by ITC [6], the thermal on-rate (kon = koff. KA) of PF4/H complexes is calculated. The short heparins bind to PF4s with ~10–20 times faster than long heparins [35].

PF4-Heparins interaction is more complex than general ligand-receptor interactions which are attributed to the electrostatic attraction. Based on special features in force-distance curves and the magnitude of PF4/H binding forces, it has been proved that long heparin bound PF4s creating additional PF4-PF4 bonds [35]. Long heparins form two types of bonds with PFs, i.e.


**Table 1.** Thermodynamic and kinetic parameters of PF4/heparin interactions [35].

(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 complexes, especially for long heparins.

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

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 of positive charges on PF4.

*3.1.1. Characteristics of human-derived HIT antibodies*

heparin. Same scale bar for all images. Adapted from [55].

bind to the PF4/H antigen.

even in the absence of heparin [53, 54].

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

**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

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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,
