AN ANTITHROMBIC MOLECULE HAVING APAC ACTIVITY FOR THE PREVENTION AND/OR TREATMENT OF THROMBOCYTOPENIA

Abstract

The invention relates to an anti-thrombotic molecule having both anti-platelet and anti-coagulant (APAC) activity and, in particular, its use as a medicament to prevent and/or treat heparin-induced thrombocytopenia (HIT) type I or II; and/or heparin-induced thrombocytopenia and thrombosis (HITT); and/or heparin-independent thrombocytopenia autoimmune HIT (aHIT); and/or vaccine-induced thrombocytopenia and thrombosis (VITT). The invention has use in both the medical and veterinary industries.

Claims

1.-22. (canceled)

23. A method for preventing and/or treating thrombocytopenia, comprising; administering to an individual to be treated, an effective amount of an antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity, wherein the antithrombotic molecule comprises a human plasma protein to which there is attached, via a plurality of linker molecules, a plurality of heparin chains each heparin chain having a MW of 10-21 KDa and wherein the number of said heparin chains attached to said plasma protein is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, thereby preventing and/or treating thrombocyptopenia.

24. The method according to claim 23, wherein said thrombocytopenia is heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; heparin-induced thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; vaccine-induced thrombocytopenia and thrombosis (VITT), or any combination thereof.

25. The method according to claim 23, wherein said APAC is administered at a dose, in blood or plasma, within the range including and between 0.15 μg/ml-10 μg/ml, including all 0.1 μg/ml there between; within the range 1 μg/ml-3 μg/ml, including all 0.1 μg/ml there between; or within the range of 0.1-0.3 mg/kg.

26.-27. (canceled)

28. The method of claim 23, wherein said thrombocytopenia is heparin-induced thrombocytopenia (HIT) type II or heparin-induced thrombocytopenia and thrombosis (HITT).

29. The method of claim 23, wherein said thrombocytopenia is immunologically-based and is heparin-induced thrombocytopenia (HIT) type II; heparin-induced thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; or is vaccine-induced thrombocytopenia and thrombosis (VITT).

30. The method of claim 23, wherein said thrombocytopenia is non-immunologically-based and is heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia and thrombosis (HITT); or is vaccine-induced thrombocytopenia and thrombosis (VITT).

31. The method of claim 23, wherein said thrombocytopenia is caused by heparin and is heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; thrombocytopenia and thrombosis (HITT); or is vaccine-induced thrombocytopenia and thrombosis (VITT).

32. The method of claim 23, wherein said antithrombotic molecule has 4, 5 or 6, heparin chains attached to said plasma protein.

33. The method of claim 23, wherein said antithrombotic molecule has 5 heparin chains attached to said plasma protein.

34. The method of claim 23, wherein said antithrombotic molecule is formulated for administration at a dose, in blood or plasma, within the range 0.15 μg/ml-10 μg/ml, including all 0.1 μg/ml there between; within the range 1 μg/ml-3 μg/ml, including all 0.1 μg/ml there between; or within the range 0.1-0.3 mg/kg.

35. The method of claim 23, wherein said human plasma protein is albumin, globulin or fibrinogen.

36. The method of claim 23, wherein said human plasma protein is serum albumin or alpha2-macroglobulin.

37. The method of claim 23, wherein said human plasma protein is recombinant.

38. The method of claim 23, wherein said plurality of heparin chains are unfractionated heparin.

39. The method of claim 23, wherein said plurality of heparin chains each have a MW of 15 KDa, 16 KDa, or 17 KDa.

40. The method of claim 23, wherein said plurality of heparin chains are recombinant.

41. The method of claim 23, wherein each linker molecule binds one molecule of heparin to said human plasma protein.

42. The method of claim 23, wherein said plurality of linker molecules are amine linkers and so links with amino groups on said heparin chains and plasma protein.

43. The method of claim 23, wherein said plurality of linker molecules conjugate with serine on the heparin chains and a lysine on the plasma protein.

44. The method of claim 23, wherein said plurality of linker molecules are hetero-bi-functional cross-linkers such as a 3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) linker or a homo-bi-functional cross-linker such as a 3,3′-Dithiodipropionicacid di(N-hydroxysuccinimide (NHS)-ester (DTSP) linker.

45. The method of claim 23, wherein said antithrombotic molecule has a coupling level (CL) of 5 heparins per human serum albumin (HSA) and the plurality of linker molecules are SPDP.

Description

[0049] The present invention will now be described by way of example only with particular reference to the following figures wherein:

[0050] FIG. 1. Shows the binding of HIT-like monoclonal Ab KKO (Arepally et al. Blood. 2000; 95:1533-1540 https://www.ncbi.nlm.nih.gov/pubmed/10688805) to PF4 in the presence and absence of UFH and APAC. A, shows the binding of HIT-like monoclonal antibody KKO (a mouse monoclonal IgG[2bkappa] antibody against the complex of human PF4 and heparin) 1) to immobilized PF4 (50 μl of 5 μg/ml per well=0.25 μg), 2) immobilized PF4 in the presence of UFH (0.1 IU/ml), and 3) immobilized PF4 in the presence of APAC (0.5, 1, 3, 10, 30, 100, 200 and 300 μg/ml; at the heparin equivalent concentration). B, shows the binding of Ab KKO 1) to immobilized PF4 (0.25 μg), 2) to immobilized PF4 in the presence of UFH (0.1 IU/ml), 3) to immobilized PF4 in the presence of both UFH (0.1 IU/ml) and APAC (3, 10, 30 and 100 μg/ml), and 4) to immobilized PF4 in the presence of APAC (3, 10, 30 and 100 μg/ml). Results are shown as mean±standard error of the mean (SEM) of 3 independent experiments.

[0051] FIG. 2. Shows the inhibitory effect of APAC on the formation of large antigenic PF4/UFH complexes. The size of the particle formation between PF4 (10 μg/ml) and UFH (0.1 IU/ml) in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5 or 10 μg/ml; informed as the heparin equivalent concentration) is displayed after 0, 1, 2, 3, 4, 5, 6 and 24 hours of incubation time. Particle formation was detected by dynamic light scattering (DLS). Results are shown as mean±standard deviation (SD) of 3 independent experiments.

[0052] FIG. 3. Shows the dissociating effect of APAC on the preformed large antigenic PF4/UFH complexes. PF4/UFH complex was first formed between 10 μg/ml of PF4 and 0.2 IU/ml of UFH. The particle size of this preformed PF4/UFH complex in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5 or 10 μg/ml; at the heparin equivalent concentration) is displayed at 0, 1, 2, 3, 4, 5, 6 and 24 hours of incubation time. Particle formation was detected by DLS. Results are shown as mean±SD of 3 independent experiments.

[0053] FIG. 4. Shows the competing effect of APAC on the formation of ultra-large immunocomplexes (ULICs) of KKO/PF4/UFH. The size of the ULICs particle formation between PF4 (10 μg/ml), UFH (0.2 IU/ml) and HIT-like monoclonal antibody KKO (30 μg) in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5 or 10 μg/ml; informed as the heparin equivalent concentration) is displayed at 0, 1, 2, 3, 4, 5, 6 and 24 hours of incubation time. Particle formation was detected by DLS. Results are shown as mean±SD of 3 independent experiments

[0054] FIG. 5. Shows the dissociating effect of APAC on the preformed ultra-large immunocomplexes (ULICs) of KKO/PF4/UFH. ULICs were first formed using PF4 (10 μg/ml), UFH (0.2 IU/ml) and HIT-like monoclonal antibody KKO (30 μg). The particle size of the preformed KKO/PF4/UFH complexes in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, or 5 μg/ml; at the heparin equivalent concentration) is displayed at 0, 1, 2, 3, 4, 5, 6 and 24 hours of incubation time. Particle formation was detected by DLS. Results are shown as mean±SD of 3 independent experiments.

[0055] FIG. 6. Shows the effect of APAC on the induction of tissue factor (TF) activity on human monocytic-like cell line (THP-1). THP-1 cells were first incubated with PF4 (10 μg/ml) and then supplemented with APAC (10, 50 or 100 μg/ml; at the heparin equivalent concentration). Control THP-1 cells were not treated with APAC. HIT-like monoclonal antibody KKO (50 μg/ml) was included to induce formation of immunocomplexes (IC). The generation of FXa activity reflected the active TF expression in the cell suspensions. Data are depicted as the mean fold-increase of the initial velocity of FXa generation relative to THP-1 cells alone. Results are shown as mean±SEM of 4 experiments.

METHODS

Conjugation

[0056] Unfractionated heparin, Hep (UFH chains were conjugated to Human Serum Albumin (HSA) through disulfide bridges created by two alternative cross-linkers and reactions routes using: [0057] i) hetero-bi-functional cross-linker 3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP). For the conjugation free amines on Ser at the Hep linker region and Lys on HSA were utilized. Hep and HSA were modified in separate reactions into sulfhydryl (—SH)—and pyridyl dithiol(-PDP)-derivatives, respectively. In the final conjugation reaction, the pyridyldithiol-group of HSA reacted with sulfhydryl group of Hep resulting in the formation of a disulphide bonded complex and the release of pyridine 2-thione. [0058] ii) homo-bi-functional cross-linker 3,3′-Dithiodipropionicacid di(N-hydroxysuccinimide (NHS)-ester) (DTSP). For the conjugation, free amines on Ser at the Hep linker region and Lys on HSA were utilized. Hep was first modified into N-hydroxysuccinimide (NHS)-ester-derivative with the release of the first NHS-group. In the final conjugation reaction the Lys of HSA reacted with the N-hydroxysuccinimide (NHS)-ester group of the derivatized Hep, resulting in the formation of a complex with a cleavable disulfide bond in the linker region and the release of the second N-hydroxy-succinimide group. [0059] The ratio of the above defined intermediate derivatives of HSA and Hep in the final conjugation reaction to produce Hep-HSA complexes is selected to yield the specified mean conjugation level (CL) in the final purified Hep-HSA complexes.

[0060] Hep-HSA complexes were purified by ultra/diafiltration and anion exchange chromatography using Q sepharose media (GE Healthcare, USA) or ultra/dialfiltration. At the end Hep-HSA complexes were eluted into phosphate buffered saline (PBS) with pH 7.4-7.5. Complexes were named as APAC- with a suffix extension designating the mean conjugation level of Hep chains to HSA. Accordingly, reference herein to a plurality of heparin chains conjugated to a human plasma protein selected from the group comprising 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, is reference to a mean conjugation level.

[0061] The general formula for APAC complexes that exemplify the invention is (Hep-NH—CO—CH.sub.2—CH.sub.2—S—S—CH.sub.2—CH.sub.2—CO—NH).sub.n-HSA where the average number of unfractionated heparin chains coupled to HSA is defined as n.

[0062] The mean conjugation level (CL) of Hep to HSA was determined using the concentration of Hep and HSA and their average molecular weights with the following equations:

mol of Hep=Hep [C]/mean Hep MW

mol of HSA=HSA [C]/HSA MW

CL=mol of Hep/mol of HSA

Hep MW=15800 or 17000

HSA MW=66472

Binding of HIT-Like Monoclonal Ab KKO to PF4 in the Presence and Absence of UFH and APAC

[0063] Immulon 4 HBX plates (Thermo Scientific, Waltham, Mass., USA) were first coated with PF4 (50 μl of 5 μg/ml in phosbate buffered saline [PBS]). In the experiment A, wells were supplemented with APAC at final concentration of 0.5, 1, 3, 10, 30, 100, 200 and 300 μg/ml. In the experiment B), APAC was supplemented at final concentration of 3, 10, 30, 100, 200 and 300 μg/ml either alone or together with constant concentration of UFH (0.1 IU/ml). PF4 alone and PF4 supplemented with UFH (0.1 IU/ml) were used as controls. Plates were incubated overnight at room temperature (RT). The next day, the wells were washed 4 times with 180 μl of PBS and the unspecific binding was blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at RT. Next, wells were supplemented with HIT-like monoclonal antibody KKO at 100 μg/ml (in 1% BSA/PBS) for 30 min at 37° C. after which wells were washed 4 times with 180 μl of PBS/0.1% Tween-20. Wells were incubated 30 min with Horse radish peroxidase (HRP) conjugated Goat Anti-Mouse IgG (Fc) 1:3000 in 1% BSA/PBS was used as the secondary Ab (Bethyl Laboratories, Montgomery, Tex., US). The wells were further washed 4 times with 180 μl PBS/0.1% Tween-20 and HRP substrate, 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Roche diagnostics, Mannheim, Germany) was added at 100 μl/well at RT to detect the secondary Ab at 405 nm and 490 nm on a BioTek Synergy 2 plate reader (BioTek Instruments Inc., Winooski, Vt., USA). Results are calculated as mean±SEM of 3 independent experiments.

Effect of APAC on Formation of Large Antigenic PF4/UFH Complexes

[0064] PF4 (10 μg/ml) was supplemented either with UFH (Hep; 0.2 IU/ml) alone, or with both UFH (Hep; 0.2 IU/ml) and APAC at increasing concentrations (0.15, 0.3, 1, 2, 3, 5 or 10 μg/ml). Particle size was measured by dynamic light scattering (DLS) immediately after addition of UFH and/or APAC and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as mean±SD of 3 independent experiments.

Effect of APAC on Dissociation of Preformed Large Antigenic Complexes

[0065] PF4 (10 μg/ml) was supplemented with UFH (0.2 IU/ml) and pre-incubated for 30 min at room temperature after which APAC was added at the increasing concentrations of 0.15, 0.3, 0.5, 1, 2, 3, and 5 μg/ml. The size of the formed particles was measured by DLS immediately and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as mean±SD of 3 independent experiments.

Effect of APAC on Formation of Ultra Large Immunocomplexes

[0066] APAC (0.15, 0.3, 1, 2, 3, or 5 μg/ml), PF4 (10 μg/ml), UFH (0.2 IU/ml) and HITT-like monoclonal antibody KKO (30 μg) were incubated together and the size of the formed ultra large immunocomplexes (ULICs) was measured by DLS immediately, and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as mean±SD of 3 independent experiments.

Effect of APAC on Disruption of Preformed Ultra Large Immunocomplexes

[0067] PF4 (10 μg/ml) was first incubated with HIT-like monoclonal Ab KKO for 5 min at RT after which, UFH (0.2 IU/ml) was added for additional 5 min to form PF4/KKO/UFH complexes. These pre-formed PF4/KKO/UFH complexes were then supplemented with APAC at 0.15, 0.3, 1, 2, 3, or 5 μg/ml. The size of the formed particles was measured by DLS immediately, and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as mean±SD of 3 independent experiments.

Effect of APAC on the Induction of FXa Activity by a Monocytic Cell Line

[0068] Tissue factor, TF production by THP-1 cells incubated with PF4/KKO±APAC. This experiment was designed to determine whether APAC would prevent the generation of tissue factor (TF) activity on human monocyte-like cells by PF4 and HIT-like monoclonal antibody KKO.

[0069] THP-1 cells (a human acute leukemia monocytic cell line) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 4.5 mg/ml glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, at 37° C. and under 5% CO.sub.2 THP-1 cells were plated on a 96-well plate at 10.sup.5 cells per well in 100 μl of RPMI-1640 supplemented with 5% FBS. THP-1 cells were incubated at 37° C. first with PF4 (10 μg/ml) for 5 min, and then for additional 30 min with APAC at final concentration of 10, 50 or 100 μg/ml. Control THP-1 cells were without APAC. In the third step, all cells were supplemented with HIT-like monoclonal Ab KKO (50 μg/ml) and incubated further overnight. Binding of KKO to cell-associated glycosaminoglycans substituted for exogenous UFH. The next day cells were washed to remove unbound ligands. FXa activity was measured using a chromogenic assay in a flat bottom 96-well plate where an aliquot (10 μl) of cell suspension was added to a mixture of Factor Vila (0.5 nM) and Factor X (160 nm) in 20 mM Tris buffer, pH 7.4 containing 100 mM NaCl and 10 mM CaCl2 for 30 min at 37° C. under 5% CO2. An activated coagulation factor FXa chromogenic substrate (0.4 mM) was added and the optical density at 405 nm was read in a kinetic mode (one read per minute) for 30 min at 37° C. The amount of FXa generated over the first 10 min was calculated relative to a standard curve using purified reagents.

[0070] The mean MW for the Hep polymer is based on the information obtained from the heparin manufacturer. HSA MW is based on ALBU_HUMAN, P02768 from UniProtKB/Swiss-Prot, isoform 1 without signal—and propeptide.

[0071] Statistics. All data are mean±SD or SEM and analyzed by SPSS for Windows, version 15.0 (SPSS Inc, Chicago, Ill.). For two-group comparison, non-parametric Mann-Whitney U test and parametric Student's t-test were applied. For multiple-group comparison, non-parametric Kruskal-Wallis test with the Dunn post hoc test and parametric ANOVA with Dunnett's correction were applied. P<0.05 was regarded as statistically significant.

Experimental Procedure

[0072] Our first objective was to determine whether APAC formed antigenic complexes with PF4 and/or whether it reduced the antigenicity of PF4/UFH complexes, defined by binding of a HIT-like antibody. Once we identified a concentration of APAC that interfered with antigenicity, our second objective was to examine the impact on the size of the large pathogenic complexes that cause HIT or HITT. Because we have found that ULICs are stable for over 24 hours, we assumed that the antigenic complex and the immune complex undergo a succession of changes over time that make it progressively more difficult to effect change. Therefore, our objective was to perform a series of experiments addressing the presumed sequence of events in the pathogenesis of HIT by asking the following 4 questions, each addressed in turn:

1) Can APAC prevent formation of PF4/UFH complexes?
2) Can APAC disrupt PF4/UFH complexes?
3) Can APAC prevent formation of ULICs? and
4) Can APAC disrupt preformed ULICs?
To do so we used a well described murine monoclonal anti-PF4/UFH antibody called KKO, and we employed dynamic light scattering (DLS) to measure the size of complexes in solution.

[0073] Lastly, we asked whether APAC would inhibit the ability of ULICs to generate active coagulation factor, FXa activity on a monocytic cell line.

Results

[0074] Note: In all experiments, the data are presented as mean±SD of at least 3 independent experiments (FIG. 2-5) or mean±SEM from 3 (FIG. 1) or 4 experiments (FIG. 6).

A. Effect of APAC on Binding of the HIT-Like Monoclonal Antibody KKO

[0075] The data shown in FIG. 1A are from ELISAs to measure the binding of the murine monoclonal HIT-like antibody to PF4/APAC. The results show that KKO binds to PF4/APAC starting at the lowest concentration tested (0.5 μg/ml) (see FIG. 1A). However, binding of KKO is lower in a dose dependent manner at higher, likely therapeutic concentrations of APAC based on subsequent results. This is the same pattern as is observed at supraoptimal concentrations of heparin attributed to formation of smaller complexes with PF4.

The Effect of APAC Added to PF4/UFH is shown in FIG. 1B, Left Side

[0076] There is a small increase in KKO binding to PF4/UFH at a concentration of APAC of 1 μg/ml, which is equivalent to binding to PF4/APAC alone, i.e. in the absence of UFH (data not shown). However, the most important effect is a dose-dependent decrease in KKO binding at all higher concentrations of APAC. Comparison of the right and left slides in FIG. 1B makes it likely that APAC dissociated large PF4/UFH complexes and binding is to, what we will propose will be small, PF4/APAC complexes. These results led to ask whether binding of PF4 to APAC generates large “pathogenic” complexes. We thought that the small size of APAC makes it unlikely to foster oligomerization of PF4 in the manner seen with UFH, and this supposition was confirmed in the experiments that are described below.

B. Effect of APAC on Formation of Large Antigenic PF4/UFH Complexes

[0077] In these and the DLS experiments that follow, PF4 (10 μg/ml) was incubated with the indicated concentration of APAC and UFH (0.2 IU/ml) as the standard starting condition. Particle size (ordinate) was measured by DLS immediately and 1, 2, 3, 4, 6, and 24 hours later (abscissa). The red line in FIG. 2 shows the absence of APAC, i.e. this is the sizes of the complexes formed between PF4 and UFH, which continue to increase in time over 24 hours incubation. In general, there is an inverse dose-dependent relationship between the concentration of APAC and size of the particles. The seemingly anomalous early result at 1 μg/ml APAC matches results of the ELISA and may represent a combination of PF4/APAC and PF4/UFH complexes or incorporation of APAC into PF4/UFH complexes. The results show that growth of complexes during 24 hours of incubation is inhibited by APAC at concentrations as low as 0.15 μg/ml and inhibition is almost complete at 3 μg/ml.

C. Effect of APAC on Dissociation of Preformed Large Antigenic Complexes

[0078] In the set of experiments shown in FIG. 3, PF4 was pre-incubated with unfractionated heparin (UFH) for 30 min at room temperature. APAC was then added at the indicated concentrations. The results show that at the lowest concentration of APAC, there is no effect on the size of the antigenic complexes. A decrease in size begins at 0.3 μg/ml and no complexes >20 nm in size is evident at the higher concentrations. A major inhibitory effect is seen at 1 μg/ml.

D. Effect of APAC on Formation of ULICs

[0079] In the experiments shown in FIG. 4, APAC was added along with PF4 (10 μg/ml), UFH (0.2 IU/ml) and KKO (30 μg) and the size of the complexes (ordinate) over time (abscissa) was measured. The data show that at low doses of APAC, immune complex formation is enhanced. This is consistent with data in previous modes showing enhanced antibody binding at formation of UFH-PF4 antigenic complexes at these concentrations. At higher concentrations, APAC totally prevented formation of ultra-large immune complexes (ULICs), again consistent with its capacity to prevent and to disrupt antigen formation. A major inhibitory effect is seen at 3 μg/ml.

E. Disruption of Preformed ULICs

[0080] This is the most stringent test, i.e. breaking up the large and stable pre-formed PF4/KKO/heparin complexes. Here, PF4 (10 μg/ml) was incubated with KKO for 5 min at RT. UFH (0.2 IU/ml) was added for 5 min at RT. Then APAC was added at increasing concentrations (0 to 5 μg/ml). The results are shown in FIG. 5. As we observed in all previous experimental conformations, low doses of APAC increased the size of the complexes. However, preformed complexes were totally disrupted at higher concentrations of APAC. A major inhibitory effect is seen at 3 μg/ml.

F. Effect of APAC on the Induction of FXa Activity by a Monocytic Cell Line

[0081] This experiment was designed to determine whether APAC would prevent the induction of tissue factor activity on THP-1 monocytic cells by PF4 and KKO. THP-1 cells were incubated with PF4 with/without APAC as follows. The order of addition was PF4 (10 μg/ml, 5 min incubation) followed by APAC (30 min) and then KKO (50 μg/ml), all at 37° C. Binding of KKO to cell-associated glycosaminoglycans substituted for exogenous UFH. After further incubation, the cells were washed to remove unbound ligands. As the measure of TF expression, an aliquot of cell suspension was detected for the amount of FXa generated. Results shown in FIG. 6 are the mean±SEM of 4 experiments, each conducted in quadruplicate wells. The results show that the higher concentrations of APAC inhibited the generation of FXa by this monocytic cell line that had been stimulated by HITT immune complexes. These results are in line with all the previous sets of experiments looking at formation/dissolution of PF4/UFH and PF4/UFH/KKO complexes.

Conclusions

[0082] These experiments support the concept that APAC provides a new approach to the treatment of HIT type II and/or HITT by combining antithrombotic activity with the capacity to interfere with ULIC formation and stability, one of the most proximal steps in the pathogenic process.

[0083] There is a successive increase in the concentration of APAC required to prevent antigen formation<dissociate antigen<prevent=dissociate immune complexes. In practical terms, administration of APAC instead of UFH would, in theory, prevent HIT from developing while higher concentrations can be used to interrupt the cycle of ULIC formation, cell activation, release of PF4 and thrombin and the feedforward prothrombotic loop that develops in these patients.

[0084] In other words, APAC should be used instead of heparin [0085] 1) Prophylactically, when there is a great suspicion of developing HIT, such as a previous history or when there is an increased risk of HIT, such as in connection with a trauma or surgical, e.g. cardiovascular, intervention and/or [0086] 2) when HIT type II or HITT has occurred then heparin needs to be stopped and APAC should replace heparin.